US20100068228A1 - Inducing Cellular Immune Responses to Hepatitis B Virus Using Peptide and Nucleic Acid Compositions - Google Patents

Inducing Cellular Immune Responses to Hepatitis B Virus Using Peptide and Nucleic Acid Compositions Download PDF

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US20100068228A1
US20100068228A1 US12/535,966 US53596609A US2010068228A1 US 20100068228 A1 US20100068228 A1 US 20100068228A1 US 53596609 A US53596609 A US 53596609A US 2010068228 A1 US2010068228 A1 US 2010068228A1
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peptide
hla
composition
hbv
peptides
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Alessandro Sette
John Sidney
Scott Southwood
Maria Vitiello
Brian Livingston
Esteban Celis
Ralph Kubo
Howard Grey
Robert Chesnut
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Epimmune Inc
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Pharmexa Inc
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Priority claimed from US08/159,339 external-priority patent/US6037135A/en
Priority claimed from US08/197,484 external-priority patent/US6419931B1/en
Priority claimed from US08/344,824 external-priority patent/US20030152580A1/en
Priority claimed from US09/189,702 external-priority patent/US7252829B1/en
Priority claimed from US09/239,043 external-priority patent/US6689363B1/en
Priority to US12/535,966 priority Critical patent/US20100068228A1/en
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12Antivirals
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503Immunoglobulin superfamily
    • C07K14/70539MHC-molecules, e.g. HLA-molecules
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00Reverse transcribing DNA viruses
    • C12N2730/00011Details
    • C12N2730/10011Hepadnaviridae
    • C12N2730/10111Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • HBV hepatitis B virus
  • cirrhosis and hepatocellular carcinoma affects at least 5% of the world's population and is a major cause of cirrhosis and hepatocellular carcinoma (Hoofnagle, J., N. Engl. J. Med. 323:337, 1990; Fields, B. and Knipe, D., In: Fields Virology 2:2137, 1990).
  • the World Health Organization lists hepatitis B as a leading cause of death worldwide, close behind chronic pulmonary disease, and more prevalent than AIDS.
  • Chronic HBV infection can range from an asymptomatic carrier state to continuous hepatocellular necrosis and inflammation, and can lead to hepatocellular carcinoma.
  • the immune response to HBV is believed to play an important role in controlling hepatitis B infection.
  • a variety of humoral and cellular responses to different regions of the HBV nucleocapsid core and surface antigens have been identified.
  • T cell mediated immunity particularly involving class I human leukocyte antigen-restricted cytotoxic T lymphocytes (CTL) is believed to be crucial in combating established HBV infection.
  • CTL cytotoxic T lymphocytes
  • HLA human leukocyte antigen
  • HLA class II restricted T cell responses are usually detected in patients with acute hepatitis, and are absent or weak in patients with chronic infection (Chisari, F. V. and Ferrari, C., Annu. Rev. Immunol. 13:29, 1995).
  • HLA Class II responses are tied to activation of helper T cells (HTLs)
  • Helper T lymphocytes which recognize Class II HLA molecules, may directly contribute to the clearance of HBV infection through the secretion of cytokines which suppress viral replication (Franco, A. et al., J. Immunol. 159:2001, 1997).
  • helper T lymphocytes which recognize Class II HLA molecules, may directly contribute to the clearance of HBV infection through the secretion of cytokines which suppress viral replication (Franco, A. et al., J. Immunol. 159:2001, 1997).
  • cytokines which suppress viral replication
  • epitope-based vaccines Upon development of appropriate technology, the use of epitope-based vaccines has several advantages over current vaccines.
  • the epitopes for inclusion in such a vaccine are to be selected from conserved regions of viral or tumor-associated antigens, in order to reduce the likelihood of escape mutants.
  • the advantage of an epitope-based approach over the use of whole antigens is that there is evidence that the immune response to whole antigens is directed largely toward variable regions of the antigen, allowing for immune escape due to mutations.
  • immunosuppressive epitopes that may be present in whole antigens can be avoided with the use of epitope-based vaccines.
  • an epitope-based vaccine approach there is an ability to combine selected epitopes (CTL and HTL) and additionally to modify the composition of the epitopes, achieving, for example, enhanced immunogenicity. Accordingly, the immune response can be modulated, as appropriate, for the target disease. Similar engineering of the response is not possible with traditional approaches.
  • epitope-based immune-stimulating vaccines Another major benefit of epitope-based immune-stimulating vaccines is their safety. The possible pathological side effects caused by infectious agents or whole protein antigens, which might have their own intrinsic biological activity, is eliminated.
  • An epitope-based vaccine also provides the ability to direct and focus an immune response to multiple selected antigens from the same pathogen. Thus, patient-by-patient variability in the immune response to a particular pathogen may be alleviated by inclusion of epitopes from multiple antigens from that pathogen in a vaccine composition.
  • a “pathogen” may be an infectious agent or a tumor associated molecule.
  • a need has existed to modulate peptide binding properties, for example so that peptides that are able to bind to multiple HLA antigens do so with an affinity that will stimulate an immune response.
  • Identification of epitopes restricted by more than one HLA allele at an affinity that correlates with immunogenicity is important to provide thorough population coverage, and to allow the elicitation of responses of sufficient vigor whereby the natural immune responses noted in self-limiting acute hepatitis, or of spontaneous clearance of chronic HBV infection is induced in a diverse segment of the population. Such a response can also target a broad array of epitopes.
  • the technology disclosed herein provides for such favored immune responses.
  • This invention applies our knowledge of the mechanisms by which antigen is recognized by T cells, for example, to develop epitope-based vaccines directed towards HBV. More specifically, this application communicates our discovery of specific epitope pharmaceutical compositions and methods of use in the prevention and treatment of HBV infection.
  • An embodiment of the present invention includes a peptide composition of less than 100 amino acid residues comprising a peptide epitope useful for inducing an immune response against hepatitis B virus (HBV) said epitope (a) having an amino acid sequence of about 8 to about 13 amino acid residues that have at least 65% identity with a native amino acid sequence for HBV, and, (b) binding to at least one MEC class I HLA allele with a dissociation constant of less than about 500 nM.
  • the peptide composition may comprise an amino acid sequence of at least 77% identity, or at least 100% identity with a native HBV amino acid sequence.
  • the peptide is one of the peptides designated as being from the envelope, polymerase, protein X, or nucleocapsid core regions of HBV. Preferred peptides are described in Tables VI through XVII or XXI.
  • An additional embodiment of the present invention comprises a composition of less than 100 amino acid residues comprising a peptide epitope useful for inducing an immune response against hepatitis B virus (HBV) said peptide (a) having an amino acid sequence of about 8 to about 13 amino acid residues and (b) bearing one of the HLA supermotifs or motifs set out in Tables I and II.
  • HBV hepatitis B virus
  • composition may comprise a peptide wherein the peptide is one of those described in Tables VI through XVII or Table XXI which bear an HLA A1, A2, A3, A24, B7, B27, B44, B58, or B62 supermotif; or an HLA A1, A3, A11, A24, or A2.1 motif or an HLA A*3301, A*3101, A*6801, B*0702, B*3501, B51, B*5301, B*5401 motif.
  • the peptide does not bear an L or M at position 2 and V at the C-terminal position 9 of a 9 amino acid peptide.
  • An alternative embodiment of the invention comprises an analog of an HBV peptide of less than 100 amino acid residues in length that bears an HLA binding motif, the analog bearing the same HLA binding motif as the peptide but comprising at least one anchor residue that is different from that of the peptide.
  • said peptide is an analog of a peptide described in Table VI through Table XVII bearing an HLA A1, A2, A3, A24, B7, B27, B44, B58, or B62 supermotif; or an HLA A1, A3, A11, A24, or A2.1 motif or A3301, A3101, A6801, B0702, B3501, B51, B5301, B5401 motif.
  • Embodiments of the invention further include a composition of less than 100 amino acid residues comprising a peptide epitope useful for inducing an immune response against hepatitis B virus (HBV) said peptide (a) having an amino acid sequence of about 9 to about 25 amino acid residues that have at least 65% identity with a native amino acid sequence for HBV and (b) binding to at least one MHC class II HLA allele with a dissociation constant of less than about 1000 nM.
  • the composition comprises a peptide that has at least 77%, or, 100% identity with a native HBV amino acid sequence.
  • the composition may comprise a peptide wherein said peptide is one of those peptides described in Table XVIII or Table XIX.
  • the invention also includes a peptide composition of less than 100 amino acid residues, said composition comprising an epitope useful for inducing an immune response against hepatitis B virus (HBV) said epitope (a) having an amino acid sequence of about 10 to about 20 amino acid residues and (b) bearing one of the class II HLA motifs set out in Table III.
  • said peptide is one of those peptides described in Table XVIII or XIX.
  • compositions that comprises an isolated nucleic acid sequence that encodes one of the peptides set out in Tables VI through XIX or XXI or XXIII.
  • an embodiment of the invention comprises a composition that comprises at least two peptides, at least one of said at least two peptides selected from Tables VI-XIX or XXI or XXIII. In a preferred embodiment, two or more of the at least two peptides are depicted in Tables VI-XIX or XXI or XXIII.
  • the composition may further comprise at least one nucleic acid sequence. In a preferred embodiment each of said at least two peptides are encoded by a nucleic acid sequence, wherein each of the nucleic acid sequences are located on a single vector.
  • Embodiments of the invention additionally include a peptide composition of less than 100 amino acid residues, said composition comprising an epitope useful for inducing an immune response against HBV, said epitope having at least one of the amino acid sequences set out in Table XXIII.
  • An alternative modality for defining the peptides in accordance with the invention is to recite the physical properties, such as length; primary, secondary and/or tertiary structure; or charge, which are correlated with binding to a particular allele-specific HLA molecule or group of allele-specific HLA molecules.
  • a further modality for defining peptides is to recite the physical properties of an HLA binding pocket, or properties shared by several allele-specific HLA binding pockets (e.g. pocket configuration and charge distribution) and reciting that the peptide fits and binds to said pocket or pockets.
  • An additional embodiment of the invention comprises a method for inducing a cytotoxic T cell response to HBV in a mammal comprising administering to said mammal at least one peptide from Tables VI to XIX or Table XXI.
  • inventions include a vaccine for treating HBV infection that induces a protective immune response, wherein said vaccine comprises at least one peptide selected from Tables VI to Table XIX or Table XXI in a pharmaceutically acceptable carrier.
  • a vaccine for preventing HBV infection that induces a protective immune response
  • said vaccine comprises at least one peptide selected from Tables VI to XIX or Table XXI in a pharmaceutically acceptable carrier.
  • the invention further includes an embodiment comprising a method for inducing a cytotoxic T cell response to HBV in a mammal, comprising administering to said mammal a nucleic acid sequence encoding a peptide selected from Tables VI to XIX or Table XXI.
  • a further embodiment of the invention comprises a kit for a vaccine for treating or preventing HBV infection, wherein the vaccine induces a protective immune response, said vaccine comprising at least one peptide selected from Tables VI to XIX or Table XXI in a pharmaceutically acceptable carrier and instructions for administration to a patient.
  • the invention includes an embodiment comprising a method for monitoring immunogenic activity of a vaccine for HBV in a patient having a known HLA-type, the method comprising incubating a T lymphocyte sample from the patient with a peptide selected from Tables VI to XIX or Table XXI which binds the product of at least one HLA allele present in said patient, and detecting for the presence of a T lymphocyte that binds to the peptide.
  • the peptide comprises a tetrameric complex.
  • FIG. 1 Illustrates the Position of Peptide Epitopes in Experimental Model Minigene Constructs
  • the peptides and corresponding nucleic acid compositions of the present invention are useful for stimulating an immune response to HBV either by stimulating the production of CTL or HTL responses.
  • the peptides which are derived directly or indirectly from native HBV amino acid sequences, are able to bind to HLA molecules and stimulate an immune response to HBV.
  • the complete polyprotein sequence from HBV and its variants can be obtained from Genbank. Peptides can also be readily determined from sequence information that may subsequently be discovered for heretofore unknown variants of HBV as will be clear from the disclosure provided below.
  • the peptides of the invention have been identified in a number of ways, as will be discussed below. Further, analog peptides have been derived and the binding activity for HLA molecules modulated by modifying specific amino acid residues to create peptide analogs exhibiting altered immunogenicity. Further, the present invention provides compositions and combinations of compositions that enable epitope-based vaccines that are capable of interacting with multiple HLA antigens to provide broader population coverage than prior vaccines.
  • Cross-reactive binding indicates that a peptide is bound by more than one HLA molecule; a synonym is degenerate binding.
  • a “cryptic epitope” elicits a response by immunization with an isolated peptide, but the response is not cross-reactive in vitro when intact whole protein which comprises the epitope is used as an antigen.
  • a “dominant epitope” is an epitope that induces an immune response upon immunization with a whole native antigen. (See, e.g., Sercarz, et al., Annu. Rev. Immunol. 11:729766 (1993)) Such a response is cross-reactive in vitro with an isolated peptide epitope.
  • an “epitope” is a set of amino acid residues which is involved in recognition by a particular immunoglobulin, or in the context of T cells, those residues necessary for recognition by T cell receptor proteins and/or Major Histocompatibility Complex (MHC) receptors.
  • MHC Major Histocompatibility Complex
  • an epitope is the collective features of a molecule, such as primary, secondary and tertiary peptide structure, and charge, that together form a site recognized by an immunoglobulin, T cell receptor or HLA molecule.
  • “high affinity” with respect to HLA class I molecules is defined as binding with an IC 50 (or K D ) of less than 50 nM. “Intermediate affinity” is binding with an IC 50 (or K D ) of between about 50 and about 500 nM. “High affinity” with respect to binding to HLA class II molecules is defined as binding with an K D of less than 100 nM. “Intermediate affinity” is binding with a K D of between about 100 and about 1000 nM. Assays for determining binding are described in detail in PCT publications WO 94/20127 and WO 94/03205. Alternatively, binding is expressed relative to a reference peptide.
  • the IC 50 's of the peptides tested may change somewhat. However, the binding relative to the reference peptide will not significantly change. For example, in an assay run under conditions such that the IC 50 of the reference peptide increases 10-fold, the IC 50 values of the test peptides will also shift approximately 10-fold. Therefore, to avoid ambiguities, the assessment of whether a peptide is a good, intermediate, weak, or negative binder is generally based on its IC 50 , relative to the IC 50 of a standard peptide.
  • HLA Human Leukocyte Antigen
  • MEC Major Histocompatibility Complex
  • HLA supertype or family describes sets of HLA molecules grouped on the basis of shared peptide-binding specificities. HLA class I molecules that share somewhat similar binding affinity for peptides bearing certain amino acid motifs are grouped into HLA supertypes.
  • HLA superfamily, HLA supertype family, and HLA xx-like supertype molecules are synonyms.
  • IC 50 is the concentration of peptide in a binding assay at which 50% inhibition of binding of a reference peptide is observed. Given the conditions in which the assays are run (i.e., limiting HLA proteins and labeled peptide concentrations), these values approximate K D values. It should be noted that IC 50 values can change, often dramatically, if the assay conditions are varied, and depending on the particular reagents used (e.g., HLA preparation, etc.). For example, excessive concentrations of HLA molecules will increase the apparent measured IC 50 of a given ligand.
  • nucleic or percent “identity,” in the context of two or more peptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using a sequence comparison algorithms or by manual alignment and visual inspection.
  • immunogenic peptide or “peptide epitope” is a peptide which comprises an allele-specific motif or supermotif such that the peptide will bind an HLA molecule and induce a CTL and/or HTL response.
  • immunogenic peptides of the invention are capable of binding to an appropriate HLA molecule and thereafter inducing a cytotoxic T cell response, or a helper T cell response, to the antigen from which the immunogenic peptide is derived.
  • isolated or “biologically pure” refer to material which is substantially or essentially free from components which normally accompany the material as it is found in its native state.
  • isolated peptides in accordance with the invention preferably do not contain materials normally associated with the peptides in their in situ environment.
  • MHC Major Histocompatibility Complex
  • motif refers to the pattern of residues in a peptide of defined length, usually a peptide of from about 8 to about 13 amino acids for a class I HLA motif and from about 6 to about 25 amino acids for a class II HLA motif, which is recognized by a particular HLA molecule.
  • Peptide motifs are typically different for each protein encoded by each human HLA allele and differ in the pattern of the primary and secondary anchor residues.
  • a “negative binding residue” is an amino acid which if present at certain positions (typically not primary anchor positions) of peptide epitope results in decreased binding affinity of the peptide for the peptide's corresponding HLA molecule.
  • peptide is used interchangeably with “oligopeptide” in the present specification to designate a series of residues, typically L-amino acids, connected one to the other, typically by peptide bonds between the amino and carboxyl groups of adjacent amino acids.
  • the preferred CTL-inducing oligopeptides of the invention are fewer than 25 residues in length, or less than 15 residues in length or 13 residues or less in length and usually consist of between about 8 and about 11 residues, preferably 9 or 10 residues.
  • the preferred HTL-inducing oligopeptides are less than about 50 residues in length and usually consist of between about 6 and about 30 residues, more usually between about 12 and 25, and often between about 15 and 20 residues.
  • “Pharmaceutically acceptable” refers to a non-toxic, inert, and physiologically compatible composition.
  • a “primary anchor residue” is an amino acid at a specific position along a peptide sequence which is understood to provide a contact point between the immunogenic peptide and the HLA molecule.
  • One to three, usually two, primary anchor residues within a peptide of defined length generally defines a “motif” for an immunogenic peptide. These residues are understood to fit in close contact with peptide binding grooves of an HLA molecule, with their side chains buried in specific pockets of the binding grooves themselves.
  • the primary anchor residues are located at position 2 (from the amino terminal position) and at the carboxyl terminal position of a 9 residue peptide in accordance with the invention.
  • the primary anchor positions for each motif and supermotif are set forth in Table I.
  • analog peptides can be created by altering the presence or absence of particular residues in these primary anchor positions. Such analogs are used to finely modulate the binding affinity of a peptide comprising a particular motif or supermotif.
  • “Promiscuous binding” is where a distinct peptide is recognized by the same T cell clone in the context of various HLA molecules.
  • a “protective immune response” refers to a CTL and/or an HTL response to an antigen from an infectious agent or a tumor antigen from which an immunogenic peptide is derived, and thereby preventing or at least partially arresting disease symptoms or progression.
  • the immune response may also include an antibody response which has been facilitated by the stimulation of helper T cells.
  • residue refers to an amino acid or amino acid mimetic incorporated into an oligopeptide by an amide bond or amide bond mimetic.
  • a “secondary anchor residue” is an amino acid at a position other than a primary anchor position in a peptide which may influence peptide binding.
  • a secondary anchor residue occurs at a significantly higher frequency amongst bound peptides than would be expected by random distribution of amino acids at one position.
  • the secondary anchor residues are said to occur at “secondary anchor positions.”
  • a secondary anchor residue can be identified as a residue which is present at a higher frequency among high affinity binding peptides, or a residue otherwise associated with high affinity binding.
  • analog peptides can be created by altering the presence or absence of particular residues in these secondary anchor positions. Such analogs are used to finely modulate the binding affinity of a peptide comprising a particular motif or supermotif.
  • a “subdominant epitope” is an epitope which evokes little or no response upon immunization with whole antigens which comprise the epitope, but for which a response can be obtained by immunization with an isolated peptide, and this response (unlike the case of cryptic epitopes) is detected when whole protein is used to recall the response in vitro or in vivo.
  • a “supermotif” is a peptide binding specificity shared by HLA molecules encoded by two or more HLA alleles. Thus, a preferably is recognized with high or intermediate affinity (as defined herein) by two or more HLA antigens.
  • Synthetic peptide refers to a peptide that is not naturally occurring, but is man-made using such methods as chemical synthesis or recombinant DNA technology.
  • each residue is generally represented by standard three letter or single letter designations.
  • the L-form of an amino acid residue is represented by a capital single letter or a capital first letter of a three-letter symbol
  • the D-form for those amino acids having D-forms is represented by a lower case single letter or a lower case three letter symbol.
  • Glycine has no asymmetric carbon atom and is simply referred to as “Gly” or G. Symbols for the amino acids are shown below.
  • a complex of an HLA molecule and a peptidic antigen acts as the ligand recognized by HLA-restricted T cells (Buus, S. et al., Cell 47:1071, 1986; Babbitt, B. P. et al., Nature 317:359, 1985; Townsend, A., and Bodmer, H., Annu. Rev. Immunol. 7:601, 1989; Germain, R. N., Annu. Rev. Immunol. 11:403, 1993).
  • HLA-peptide complexes Furthermore, x-ray crystallographic analysis of HLA-peptide complexes has revealed pockets within the peptide binding cleft of HLA molecules which accommodate allele-specific residues borne by peptide ligands; these residues in turn determine the HLA binding capacity of the peptides in which they are present (Brown, J. H. et al., Nature 364:33, 1993; Guo, H. C. et al., Proc. Natl. Acad. Sci. USA 90:8053, 1993; Guo, H. C. et al., Nature 360:364, 1992; Silver, M. L. et al., Nature 360:367, 1992; Matsumura, M.
  • class I and class II allele-specific HLA binding motifs or class I supermotifs allows identification of regions within a protein that have the potential of binding particular HLA antigens (see also e.g., Sette, A. and Grey, H. M., Curr. Opin. Immunol. 4:79, 1992; Sinigaglia, F. and Hammer, J., Curr. Biol. 6:52, 1994; Engelhard, V. H., Curr. Opin. Immunol. 6:13, 1994 Kast, W. M. et al., J. Immunol., 152:3904, 1994).
  • recall responses were detected by culturing PBL from subjects that had been naturally exposed to the antigen, for instance through infection, and thus had generated an immune response “naturally”.
  • PBL from subjects were cultured in vitro for 1-2 weeks in the presence of test peptide plus antigen presenting cells (APC) to allow activation of “memory” T cells, as compared to “naive” T-cells.
  • APC antigen presenting cells
  • T cell activity is detected using assays for T cell activity including 51 Cr release involving peptide-sensitized targets, T cell proliferation or lymphokine release.
  • epitope selection encompassing identification of peptides capable of binding at high or intermediate affinity to multiple HLA molecules is preferably utilized, most preferably these epitopes bind at high or intermediate affinity to two or more allele specific HLA molecules.
  • CTL-inducing peptides of interest for vaccine compositions preferably include those that have a binding affinity for class I HLA molecules of less than 500 nM.
  • HTL-inducing peptides preferably include those that have a binding affinity for class II HLA molecules of less than 1000 nM.
  • peptide binding is assessed by testing the capacity of a candidate peptide to bind to a purified HLA molecule in vitro. Peptides exhibiting high or intermediate affinity are then considered for further analysis. Selected peptides are tested on other members of the supertype family.
  • peptides that exhibit cross-reactive binding preferably are then used in cellular screening analyses.
  • a peptide is considered to be an epitope if it possesses the molecular features that form the binding site for a particular immunoglobulin or T cell receptor protein.
  • High HLA binding affinity is correlated with greater immunogenicity. Greater immunogenicity can be manifested in several different ways. Immunogenicity corresponds to whether an immune response is elicited at all, and to the vigor of any particular response. For example, a peptide might elicit an immune response in a diverse array of the population, yet in no instance produce a vigorous response. In accordance with these principles, close to 90% of high binding peptides have been found to be immunogenic, as contrasted with about 50% of the peptides which bind with intermediate affinity. Moreover, higher binding affinity peptides leads to more vigorous immunogenic responses. As a result, less peptide is required to elicit a similar biological effect if a high affinity binding peptide is used. Thus, in preferred embodiments of the invention, high binding epitopes are particularly desired.
  • HBV hepatitis B virus
  • the binding affinity of peptides for HLA molecules can be determined as described in Example 1, below.
  • residues 9, 45, 63, 66, 67, 70, and 99 were considered to make up the B pocket, and to determine the specificity for the residue in the second position of peptide ligands.
  • residues 77, 80, 81, and 116 were considered to determine the specificity of the F pocket, and to determine the specificity for the C-terminal residue of a peptide ligand bound by the HLA molecule.
  • Peptides of the present invention may also include epitopes that bind to MHC class II DR molecules.
  • a significant difference between class I and class II HLA molecules is that, although a stringent size restriction exists for peptide binding to class I molecules, a greater degree of heterogeneity in both sizes and binding frame positions of the motif, relative to the N and C termini of the peptide, can be demonstrated for class II peptide ligands. This increased heterogeneity is due to the structure of the class II-binding groove which, unlike its class I counterpart, is open at both ends. Crystallographic analysis of DRB*0101-peptide complexes (see, e.g., Madden, D. R. Ann. Rev. Immunol.
  • peptides of the present invention are identified by any one of several HLA-specific amino acid motifs. If the presence of the motif corresponds to the ability to bind several allele-specific HLA antigens it is referred to as a supermotif.
  • the allele-specific HLA molecules that bind to peptides that possess a particular amino acid supermotif are collectively referred to as an HLA “supertype.”
  • peptide motifs and supermotifs described below provide guidance for the identification and use of peptides in accordance with the invention.
  • Examples of peptide epitopes bearing the respective supermotif or motif are included in Tables as designated in the description of each motif or supermotif.
  • the IC 50 values of standard peptides used to determine binding affinities for Class I peptides are shown in Table IV.
  • the IC 50 values of standard peptides used to determine binding affinities for Class II peptides are shown in Table V.
  • the peptides used as standards for the binding assay are examples of standards; alternative standard peptides can also be used when performing such an analysis.
  • peptide epitope sequences listed in each Table protein sequence data from twenty HBV strains (HPBADR, HPBADR1CG, HPBADRA, HPBADRC, HPBADRCG, HPBCGADR, HPBVADRM, HPBADW, HPBADW1, HPBADW2, HPBADW3, HPBADWZ, HPBHEPB, HPBVADW2, HPBADR, HPBV, HPBVAYWC, HPBVAYWCI, NAD HPBVAYWE) were evaluated for the presence of the designated supermotif or motif.
  • Peptide epitopes were also selected on the basis of their conservancy. A criterion for conservancy requires that the entire sequence of a peptide be totally conserved in 75% of the sequences available for a specific protein.
  • the percent conservancy of the selected peptide epitopes is indicated on the Tables.
  • the frequency i.e. the number of strains of the 20 strains in which the peptide sequence was identified, is also shown.
  • the “1 st position” column in the Tables designates the amino acid position of the HBV polyprotein that corresponds to the first amino acid residue of the epitope. Preferred peptides are designated by an asterisk.
  • the HLA-A1 supermotif is characterized by peptides having a general motif of small (T or S) and hydrophobic (L, I, V, M, or F) primary anchor residues in position 2, and aromatic (Y, F, or W) primary anchor residues at the C-terminal position
  • the corresponding family of HLA molecules that bind to the A1 supermotif includes A*0101, A*2601, A*2602, A*2501, and A*3201.
  • the HLA-A2 supermotif is characterized by the presence in peptide ligands of small or aliphatic amino acids (L, I, V, M, A, T, or Q) at position 2 and L, I, V, M, A, or T at the C-terminal position. These positions are referred to as primary anchors.
  • the corresponding family of HLA molecules (the HLA-A2 supertype that binds these peptides) is comprised of at least nine HLA-A proteins: A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*0207, A*6802, and A*6901.
  • binding to each of the individual allele-specific HLA molecules can be modulated by substitutions at the primary anchor and/or secondary anchor positions.
  • the HLA-A3 supermotif is characterized by peptide ligands having primary anchor residues: A, L, I, V, M, S, or, T at position 2, and positively charged residues, such as R or K at the C-terminal position (in position 9 of 9-mers).
  • Exemplary members of the corresponding HLA family of HLA molecules (the HLA-A3 superfamily) that bind the A3 supermotif include: A3 (A*0301), A11 (A*1101), A31 (A*3101), A*3301, and A*6801.
  • Other allele-encoded HLA molecules predicted to be members of the A3 superfamily include A34, A66, and A*7401.
  • peptide binding to each of the individual allele-specific HLA proteins can be modulated by substitutions of amino acids at the primary and/or secondary anchor positions of the peptide.
  • the HLA-A24 supermotif is characterized by the presence in peptide ligands of an aromatic (F, W, or Y) residue as a primary anchor in position 2 and a hydrophobic (Y, F, L, I, V, or M) residue as primary anchor at the C-terminal position.
  • the corresponding family of HLA molecules that bind to the A24 supermotif includes A*2402, A*3001, and A*2301. Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary anchor positions.
  • the HLA-B7 supermotif is characterized by peptides bearing proline in position 2 as a primary anchor and hydrophobic or aliphatic amino acids (L, I, V, M, A, F, W, or Y) as the primary anchor at the C-terminal position.
  • the corresponding family of HLA molecules that bind the B7 supermotif (the HLA-B7 supertype) is comprised of at least a dozen HLA-B proteins including B7, B*3501-1, B*3502-2, B*3501-3, B51, B*5301, B*5401, B*5501, B*5401, B*5501, B*5502, B*5601, B*6701, and B*7801 (See, e.g., Sidney, et al., J. Immunol. 154:247 (1995); Barber, et al., Curr. Biol.
  • peptide binding to each of the individual allele-specific HLA proteins can be modulated by substitutions at the primary and/or secondary anchor positions of the peptide.
  • the HLA-B27 supermotif is characterized by the presence in peptide ligands of positively charged (R, H, or K) residues as primary anchors at position 2 and hydrophobic (A, L, I, V, M, Y, F, or W) residues as primary anchors at the C-terminal.
  • Exemplary members of the corresponding HLA molecules that bind to the B27 supermotif (the B27 supertype) include B*14, B*1509, B*38, B*3901, B*3902, B*73, and various B27 subtypes.
  • Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary anchor positions.
  • the HLA-B44 supermotif is characterized by the presence in peptide ligands of negatively charged (D or E) residues as a primary anchor in position 2, and hydrophobic residues (F, W, Y, L, I, M V, or A) as a primary anchor at the C-terminal.
  • exemplary members of the corresponding family of HLA molecules that bind to the B44 supermotif (the B44 supertype) include B*3701, B*4402, B*4403, B60, and B61. Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary anchor positions.
  • the HLA-B58 supermotif is characterized by the presence in peptide ligands of small aliphatic residues (A, S, or T) as primary anchor residues at position 2 and aromatic or hydrophobic residues (F, W, Y, L, I, or V) as primary anchor residues at the C-terminal.
  • Exemplary members of the corresponding HLA molecules that bind to the B58 supermotif (the B58 supertype) include B*1516, B*1517, B*5701, B*5702, and B*58.
  • Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary anchor positions.
  • the HLA-B62 supermotif is characterized by the presence in peptide ligands of the polar aliphatic residue Q or the hydrophobic aliphatic residues (L, V, M, or I) as a primary anchor in position 2 and hydrophobic residues (F, W, Y, M, I, or V) as a primary anchor at the C-terminal position.
  • Exemplary members of the corresponding HLA molecules that a bind to the B62 supermotif include B46, B52, B62, B75, and B77. Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary anchor positions.
  • the allele-specific HLA-A1 motif is characterized by the presence in peptide ligands of T, S, or M as a primary anchor residue at position 2 and the presence of Y as a primary anchor residue at the C-terminal position.
  • a primary anchor residue may be present at position 3 rather than position 2.
  • This motif is characterized by the presence of D, E, A, or S as a primary anchor residue in position 3 and a Y as a primary anchor residue at the C-terminus.
  • Peptide binding to HLA A1 can be modulated by substitutions at primary and/or secondary anchor positions.
  • Representative peptide epitopes that contain the A1 motif are set forth on the attached Table XV.
  • the allele-specific HLA-A3 motif is characterized by the presence in peptide ligands of L, M, V, I, S, A, T, F, C, G, or D as a primary anchor residue at position 2 and the presence of K, Y, R, H, F, or A as the primary anchor residue at the C-terminal position.
  • Peptide binding to HLA-A3 can be modulated by substitutions at primary and/or secondary anchor positions.
  • the allele-specific HLA-A11 motif is characterized by the presence in peptide ligands of V, T, M, L, I, S, A, G, N, C, D, or F as a primary anchor residue in position 2 and K, R, Y, or H as a primary anchor residue at the C-terminal position.
  • Peptide binding to HLA-A11 can be modulated by substitutions at primary and/or secondary anchor positions.
  • peptide epitopes that contain the A11 motif are set forth on the attached Table XVI; peptides bearing the A3 allele-specific motif are also present in Table XVII.
  • the A11 and A3 motifs have a number of anchor residues in common, separate tables would provide a number of redundant entries.
  • the allele-specific HLA-A24 motif is characterized by the presence in peptide ligands of Y, F, W, or M as a primary anchor residue in position 2 and F, L, I, or W as a primary anchor residue at the C-terminal position.
  • Peptide binding to HLA-A24 molecules can be modulated by substitutions at primary and/or secondary anchor positions.
  • the allele-specific HLA-A2.1 motif was first determined to be characterized by the presence in peptide ligands of L, M, V, I, A or T as a primary anchor residue in position 2 and, L, V, I, A, or T as a primary anchor residue at the C-terminal position.
  • the preferred and tolerated residues that characterize the primary anchor positions of the HLA-A2.1 motif are identical to the preferred residue of the A2 supermotif.
  • Secondary anchor residues that characterize the A2.1 motif have additionally been defined as disclosed herein. These are disclosed in Table II. Peptide binding to HLA-A2.1 molecules can be modulated by substitutions at primary and/or secondary anchor positions.
  • peptide epitopes that contain the A2.1 motif are set forth on the attached Table VII. These peptides, which bear the HLA-A2 supermotif, also contain secondary anchor residues that are characteristic of the HLA-A2.1 motif. In one embodiment, the peptide epitope does not bear an L or M at position 2 and V at the C-terminal position 9 of a 9-amino acid peptide.
  • HLA DRB1*0401 Motifs have also been identified for peptides that bind to three common HLA class II types, HLA DRB1*0401, DRB1*0101, and DRB1*0701.
  • Peptides binding to these DR molecules carry a motif characterized by a large aromatic or hydrophobic residue in position 1 (Y, F, W, L, I, V, or M) and a small, non-charged residue in position 6 (S, T, C, AP, V, I, L, or M). Allele specific secondary effects and secondary anchors for each of these HLA types have also been identified. These are set forth in Table III.
  • Peptide binding to HLA-DR4, DR1, and DR7 can be modulated by substitutions at primary and/or secondary anchor positions.
  • Two alternative motifs characterize peptides that bind to HLA-DR3 molecules.
  • a large, hydrophobic residue (I, L, V, M, Y, or F) is present in anchor position 1 and D is present as an anchor at position 4, which is defined as being 3 positions from anchor position 1 towards the carboxyl terminus regardless of the location of anchor position 1 in the peptide.
  • Lack of either the large, hydrophobic residue at anchor position 1, or of the negatively charged or amide-like anchor residue at position 4 may be compensated for by the presence of a positive charge at position 6 (which is defined as being 5 positions from anchor position 1 towards the carboxyl terminus).
  • Vaccines that have broad population coverage are preferred because they are more commercially viable and generally applicable to the most people. Broad population coverage can be obtained using the peptides of the invention (and nucleic acid compositions that encode such peptides) through selecting peptide epitopes that bind to HLA alleles which, when considered in total, are present in most of the population. Table XX lists the overall frequencies of the A2-, A3-, and B7-supertypes in various ethnicities. Coverage in excess of 80% is achieved with these motifs. These results suggest that effective and non-ethnically biased population coverage is achieved upon use of a limited number of cross-reactive peptides. Although the population coverage reached with these three main peptide specificities is high, coverage can be expanded to reach 95% population coverage and above, and more easily achieve truly multispecific responses upon use of additional supermotif or allele-specific motif bearing peptides.
  • Table XX summarizes the HLA supertypes that have been identified, and indicates an estimate of their combined prevalence in major ethnic groups.
  • the B44-, A1-, and A24-supertypes are present, on average, in over 25% of the world's major ethnic populations. While less prevalent overall, the B27-, B58-, and B62 supertypes are each present with a frequency >25% in at least one major ethnic group.
  • the Table indicates the population coverage achieved by the A2-, A3-, and B7-supertypes, and the incremental coverage obtained by the addition of A1-, A24-, and B44-supertypes, or all of the supertypes described herein. As shown, by including epitopes from the six most frequent supertypes, an average population coverage of 99% is obtained for five major ethnic groups.
  • peptides with suitable cross-reactivity among all alleles of a superfamily are identified by the screening procedures described above, cross-reactivity is not always complete and in such cases procedures to further increase cross-reactivity of peptides can be useful; such procedures can also be used to modify other properties of the peptides. Having established the general rules that govern cross-reactivity of peptides for HLA alleles within a given motif or supermotif, modification (i.e., analoging) of the structure of peptides of particular interest in order to achieve broader (or otherwise modified) HLA binding capacity can be performed.
  • peptides which exhibit the broadest cross-reactivity patterns can be produced in accordance with the teachings herein.
  • the strategy employed utilizes the motifs or supermotifs which correlate with binding to certain HLA molecules.
  • the motifs or supermotifs are defined by having primary anchors, though secondary anchors can also be modified.
  • Analog peptides can be created by substituting amino acids residues at primary anchor, secondary anchor, or at primary and secondary anchor positions. Generally, analogs are made for peptides that already bear a motif or supermotif.
  • Preferred secondary anchor residues of supermotifs and motifs that have been defined for HLA class I and class II binding peptides are shown in Tables II and III, respectively.
  • residues are defined which are deleterious to binding to allele-specific HLA molecules or members of HLA supertypes that bind to the respective motif or supermotif (Tables II and III). Accordingly, removal of residues that are detrimental to binding can be performed in accordance with the present invention.
  • the incidence of cross-reactivity increases from 22% to 37% (see, e.g., Sidney, J. et al., Hu. Immunol. 45:79, 1996).
  • one strategy to improve the cross-reactivity of peptides within a given supermotif is simply to delete one or more of the deleterious residues present within a peptide and substitute a small “neutral” residue such as Ala (that may not influence T cell recognition of the peptide).
  • An enhanced likelihood of cross-reactivity is expected if, together with elimination of detrimental residues within a peptide, residues associated with high affinity binding to multiple alleles within a superfamily are inserted.
  • the variant peptide may be used to immunize T cells in vitro from individuals of the appropriate HLA allele, and the cells' capacity to induce lysis of wild type peptide sensitized target cells is evaluated.
  • targets cells that have been either infected or transfected with the appropriate genes to establish whether endogenously produced antigen is also recognized by the relevant T cells.
  • Another embodiment of the invention to ensure adequate numbers of cross-reactive cellular binders is to create analogs of weak binding peptides.
  • Class I peptides exhibiting binding affinities of 500-50000 nM, and carrying an acceptable but suboptimal primary anchor residue at one or both positions can be “fixed” by substituting preferred anchor residues in accordance with the respective supertype.
  • the analog peptides can then be tested for crossbinding activity.
  • Another embodiment for generating effective peptide analogs involves the substitution of residues that have an adverse impact on peptide stability or solubility in a liquid environment. This substitution may occur at any position of the peptide epitope.
  • a cysteine (C) can be substituted out in favor of ⁇ -amino butyric acid. Due to its chemical nature, cysteine has the propensity to form disulfide bridges and sufficiently alter the peptide structurally so as to reduce binding capacity. Substituting ⁇ -amino butyric acid for C not only alleviates this problem, but actually improves binding and crossbinding capability in certain instances (Review: A. Sette et al, In: Persistent Viral Infections, Eds. R.
  • CTL and HTL responses are not directed against all possible epitopes. Rather, they are restricted to a few immunodominant determinants (Zinkernagel, et al., Adv. Immunol. 27:5159, 1979; Bennink, et al., J. Exp. Med. 168:19351939, 1988; Rawle, et al., J. Immunol. 146:3977-3984, 1991).
  • dominance and subdominance are relevant to immunotherapy of both infectious diseases and cancer.
  • recruitment of subdominant epitopes can be important for successful clearance of the infection, especially if dominant CTL or HTL specificities have been inactivated by functional tolerance, suppression, mutation of viruses and other mechanisms (Franco, et al., Curr. Opin. Immunol. 7:524-531, (1995)).
  • CTLs recognizing at least some of the highest binding affinity peptides might be functionally inactivated. Lower binding affinity peptides are preferentially recognized at these times.
  • TAA tumor infiltrating lymphocytes
  • CTL tumor infiltrating lymphocytes
  • T cells to dominant epitopes may have been clonally deleted, selecting subdominant epitopes may allow extant T cells to be recruited, which will then lead to a therapeutic response.
  • the binding of HLA molecules to subdominant epitopes is often less vigorous than to dominant ones. Accordingly, there is a need to be able to modulate the binding affinity of particular immunogenic epitopes for one or more HLA molecules, and thereby to modulate the immune response elicited by the peptide.
  • analog peptides which elicit a more vigorous response This ability would greatly enhance the usefulness of peptide-based vaccines and therapeutic agents.
  • Computer programs that allow the rapid screening of protein sequences for the occurrence of the subject supermotifs or motifs are encompassed by the present invention; as are programs that permit the generation of analog peptides. These programs are implemented to analyze any identified amino acid sequence or operate on an unknown sequence and simultaneously determine the sequence and identify motif-bearing epitopes thereof; analogs can be simultaneously determined as well.
  • the identified sequences will be from a pathogenic organism or a tumor-associated peptide.
  • the target molecules considered herein include all of the HBV proteins (e.g. surface, core, polymerase, and X).
  • peptides are also selected on the basis of their conservancy.
  • a presently preferred criterion for conservancy defines that the entire sequence of a peptide be totally conserved in 75% of the sequences evaluated for a specific protein; this definition of conservancy has been employed herein.
  • ⁇ G a 1i ⁇ a 2i ⁇ a 3i . . . ⁇ a ni
  • a ij is a coefficient that represents the effect of the presence of a given amino acid (j) at a given position (i) along the sequence of a peptide of n amino acids.
  • all protein sequence or translated sequence may be analyzed using software developed to search for motifs, for example the “FINDPATTERNS” program (Devereux, et al. Nucl. Acids Res. 12:387-395, 1984) or MotifSearch 1.4 software program (D. Brown, San Diego, Calif.) to identify potential peptide sequences containing appropriate HLA binding motifs.
  • motifs for example the “FINDPATTERNS” program (Devereux, et al. Nucl. Acids Res. 12:387-395, 1984) or MotifSearch 1.4 software program (D. Brown, San Diego, Calif.) to identify potential peptide sequences containing appropriate HLA binding motifs.
  • FINDPATTERNS Disevereux, et al. Nucl. Acids Res. 12:387-395, 1984
  • MotifSearch 1.4 software program D. Brown, San Diego, Calif.
  • HBV peptides and analogs thereof that are able to bind HLA supertype groups or allele-specific HLA molecules have been identified (Tables VI-XIX; Table XXI).
  • HLA binding peptides Once HLA binding peptides are identified, they can be tested for the ability to elicit a T-cell response.
  • the preparation and evaluation of motif-bearing peptides are described in PCT publications WO 94/20127 and WO 94/03205. Briefly, peptides comprising epitopes from a particular antigen are synthesized and tested for their ability to bind to the appropriate HLA proteins in assays using, for example, purified HLA class I molecules and radioiodonated peptides and/or cells expressing empty class I molecules (which lack peptide in their receptor) by, for instance, immunofluorescent staining and flow microfluorimetry, peptide-dependent class I assembly assays, and inhibition of CTL recognition by peptide competition.
  • Those peptides that bind to the class I molecule are further evaluated for their ability to serve as targets for CTLs derived from infected or immunized individuals, as well as for their capacity to induce primary in vitro or in vivo CTL responses that can give rise to CTL populations capable of reacting with selected target cells associated with a disease.
  • Corresponding assays are used for evaluation of HLA class II binding peptides.
  • Conventional assays utilized to detect CTL responses include proliferation assays, lymphokine secretion assays, direct cytotoxicity assays, and limiting dilution assays.
  • antigen-presenting cells that have been incubated with a peptide can be assayed for the ability to induce CTL responses in responder cell populations.
  • Antigen-presenting cells can be normal cells such as peripheral blood mononuclear cells or dendritic cells.
  • mutant mammalian cell lines that are deficient in their ability to load class I molecules with internally processed peptides and that have been transfected with the appropriate human class I gene may be used to test for the capacity of the peptide to induce in vitro primary CTL responses.
  • Peripheral blood lymphocytes may be used as the responder cell source of CTL precursors.
  • the appropriate antigen-presenting cells are incubated with peptide and the peptide-loaded antigen-presenting cells are then incubated with the responder cell population under optimized culture conditions.
  • Positive CTL activation can be determined by assaying the culture for the presence of CTLs that kill radio-labeled target cells, both specific peptide-pulsed targets as well as target cells expressing endogenously processed forms of the HBV antigen from which the peptide sequence was derived.
  • HTL activation may also be assessed using such techniques as T cell proliferation and secretion of lymphokines, e.g. IL-2.
  • lymphokines e.g. IL-2.
  • HLA transgenic mice can be used to determine immunogenicity of peptide epitopes.
  • transgenic mouse models including mice with human A2.1, A11, and B7 alleles have been characterized and others (e.g., transgenic mice for HLA-A1 and A24) are being developed.
  • HLA-DR1 and HLA-DR3 mouse models have also been developed. Additional transgenic mouse models with other HLA alleles may be generated as necessary.
  • Mice may be immunized with peptides emulsified in Incomplete Freund's Adjuvant and the resulting T cells tested for their capacity to recognize peptide-pulsed target cells and target cells transfected with appropriate genes.
  • CTL responses may be analyzed using cytotoxicity assays described above.
  • HTL responses may be analyzed using such assays as T cell proliferation or secretion of lymphokines.
  • Peptides in accordance with the invention can be prepared synthetically, by recombinant DNA technology, or from natural sources such as native tumors or pathogenic organisms.
  • Peptide epitopes may be synthesized individually or as polyepitopic peptides.
  • the peptide will preferably be substantially free of other naturally occurring host cell proteins and fragments thereof, in some embodiments the peptides may be synthetically conjugated to native fragments or particles.
  • the peptides in accordance with the invention can be a variety of lengths, and either in their neutral (uncharged) forms or in forms which are salts. Peptides may be synthesized The peptides in accordance with the invention are either free of modifications such as glycosylation, side chain oxidation, or phosphorylation; or they contain these modifications, subject to the condition that modifications do not destroy the biological activity of the peptides as described herein.
  • the peptide will be as small as possible while still maintaining substantially all of the biological activity of the large peptide.
  • HLA class II binding peptides may be optimized to a length of about 6 to about 25 amino acids in length, preferably to between about 13 and about 20 residues.
  • the peptides are commensurate in size with endogenously processed pathogen-derived peptides or tumor cell peptides that are bound to the relevant HLA molecules.
  • peptides of other lengths can be carried out using the techniques described herein (e.g., the disclosures regarding primary and secondary anchor positions).
  • epitopes can be present in a frame-shifted manner, e.g. a 10 amino acid long peptide could contain two 9 amino acid long epitopes and one 10 amino acid long epitope; each epitope can be exposed and bound by an HLA molecule upon administration of a plurality of such peptides.
  • This larger, preferably multi-epitopic, peptide can then be generated synthetically, recombinantly, or via cleavage from the native source.
  • the peptides of the invention can be prepared in a wide variety of ways.
  • the peptides can be synthesized in solution or on a solid support in accordance with conventional techniques.
  • Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart & Young, S OLID P HASE P EPTIDE S YNTHESIS, 2 D. ED ., Pierce Chemical Co. (1984). Further, individual peptides may be joined using chemical ligation to produce larger peptides.
  • recombinant DNA technology may be employed wherein a nucleotide sequence which encodes an immunogenic peptide of interest is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression.
  • a nucleotide sequence which encodes an immunogenic peptide of interest is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression.
  • These procedures are generally known in the art, as described generally in Sambrook et al., M OLECULAR C LONING, A L ABORATORY M ANUAL , Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989).
  • recombinant polypeptides which comprise one or more peptide sequences of the invention can be used to present the appropriate T cell epitope.
  • nucleotide coding sequence for peptides of the preferred lengths contemplated herein can be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci, et al., J. Am. Chem. Soc. 103:3185 (1981) modification can be made simply by substituting the appropriate and desired nucleic acid base(s) for those that encode the native peptide sequence.
  • the coding sequence can then be provided with appropriate linkers and ligated into expression vectors commonly available in the art, and the vectors used to transform suitable hosts to produce the desired fusion protein. A number of such vectors and suitable host systems are now available.
  • the coding sequence will be provided with operably linked start and stop codons, promoter and terminator regions and usually a replication system to provide an expression vector for expression in the desired cellular host.
  • promoter sequences compatible with bacterial hosts are provided in plasmids containing convenient restriction sites for insertion of the desired coding sequence.
  • the resulting expression vectors are transformed into suitable bacterial hosts.
  • yeast, insect or mammalian cell hosts may also be used, employing suitable vectors and control sequences.
  • HLA class I and class II binding peptides as described herein can be used, in one embodiment of the invention, as reagents to evaluate an immune response.
  • the immune response to be evaluated may be induced by using as an immunogen any agent that would potentially result in the production of antigen-specific CTLs or HTLs to the peptide epitope(s) to be employed as the reagent.
  • the peptide reagent is not used as the immunogen.
  • a peptide of the invention may be used in a tetramer staining assay to assess peripheral blood mononuclear cells for the presence of antigen-specific CTLs following exposure to a pathogen or immunogen.
  • the HLA-tetrameric complex is used to directly visualize antigen-specific CTLs (see, e.g., Ogg et al. Science 279:2103-2106, 1998; and Altman et al. Science 174:94-96, 1996) and determine the frequency of the antigen-specific CTL population in a sample of peripheral blood mononuclear cells.
  • a tetramer reagent using a peptide of the invention may be generated as follows: A peptide that binds to an allele-specific HLA molecules, or supertype molecules, is refolded in the presence of the corresponding HLA heavy chain and 132-microglobulin to generate a trimolecular complex. The complex is biotinylated at the carboxyl terminal end of the heavy chain at a site that was previously engineered into the protein. Tetramer formation is then induced by the addition of streptavidin. By means of fluorescently labeled streptavidin, the tetramer can be used to stain antigen-specific cells. The cells may then be identified, for example, by flow cytometry. Such an analysis may be used for diagnostic or prognostic purposes.
  • Peptides of the invention may also be used as reagents to evaluate immune recall responses.
  • patient PBC samples from individuals with acute hepatitis B or who have recently recovered from acute hepatitis B may be analyzed for the presence of HBV antigen-specific CTLs using HBV-specific peptides.
  • a blood sample containing mononuclear cells may be evaluated by cultivating the PBCs and stimulating the cells with a peptide of the invention. After an appropriate cultivation period, the expanded cell population may be analyzed for cytotoxic activity.
  • the peptides may also be used as reagents to evaluate the efficacy of a vaccine.
  • PBMCs obtained from a patient vaccinated with an immunogen may be analyzed using, for example, either of the methods described above.
  • a patient is HLA typed, and appropriate peptide reagents that recognize allele-specific molecules present in that patient may be selected for the analysis.
  • the immunogenicity of the vaccine will be indicated by the presence of HBV epitope-specific CTLs in the PBMC sample.
  • Vaccines that contain as an active ingredient an immunogenically effective amount of one or more peptides as described herein are a further embodiment of the invention.
  • vaccine compositions.
  • Such vaccine compositions can include, for example, lipopeptides (Vitiello, A. et al., J. Clin. Invest. 95:341, 1995), peptides compositions encapsulated in poly(DL-lactide-co-glycolide) (PLG) microspheres (see, e.g., Eldridge, et al. Molec. Immunol. 28:287-294, 1991: Alonso et al.
  • ICOMS immune stimulating complexes
  • MAPs multiple antigen peptide systems
  • vaccines in accordance with the invention encompass compositions of one or more of the claimed peptide(s) that can be introduced into a host, including humans, linked to its own carrier, or as a homopolymer or heteropolymer of active peptide units.
  • a polymer has the advantage of increased immunological reaction and, where different peptides are used to make up the polymer, the additional ability to induce antibodies and/or CTLs that react with different antigenic determinants of the pathogenic organism or tumor-related peptide targeted for an immune response.
  • useful carriers that can be used with vaccines of the invention are well known in the art, and include, e.g., thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly L-lysine, poly L-glutamic acid, influenza, hepatitis B virus core protein, hepatitis B virus recombinant vaccine and the like.
  • the vaccines can contain a physiologically tolerable (i.e., acceptable) diluent such as water, or saline, preferably phosphate buffered saline.
  • the vaccines also typically include an adjuvant.
  • Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are examples of materials well known in the art. Additionally, as disclosed herein, CTL responses can be primed by conjugating peptides of the invention to lipids, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P 3 CSS).
  • P 3 CSS tripalmitoyl-S-glycerylcysteinlyseryl-serine
  • the immune system of the host upon immunization with a peptide composition in accordance with the invention, via injection, aerosol, oral, transdermal, transmucosal, intrapleural, intrathecal, or other suitable routes, the immune system of the host responds to the vaccine by producing large amounts of CTLs specific for the desired antigen, and the host becomes at least partially immune to later infection, or at least partially resistant to developing an ongoing chronic infection.
  • a preferred embodiment of such a composition comprises class I and class II epitopes in accordance with the invention.
  • An alternative embodiment of such a composition comprises a class I and/or class II epitope in accordance with the invention, along with a PADRE® (Epimmune, San Diego, Calif.) molecule (described in the related U.S. Ser. No. 08/485,218, which is a CIP of U.S. Ser. No. 08/305,871, now U.S. Pat. No. 5,736,142, which is a CIP of abandoned application U.S. Ser. No. 08/121,101.)
  • PADRE® Epimmune, San Diego, Calif.
  • any of these embodiments can be administered as a nucleic acid mediated modality.
  • the peptides of the invention can also be expressed by viral or bacterial vectors.
  • expression vectors include attenuated viral hosts, such as vaccinia or fowlpox. This approach involves the use of vaccinia virus as a vector to express nucleotide sequences that encode the peptides of the invention. Upon introduction into an acutely or chronically infected host or into a non-infected host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits a host CTL and/or HTL response.
  • Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. No.
  • BCG Bacillus Calmette Guerin
  • BCG vectors are described in Stover, et al. Nature 351:456-460 (1991).
  • a wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention e.g. adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like, will be apparent to those skilled in the art from the description herein.
  • Antigenic peptides are used to elicit a CTL and/or HTL response ex vivo, as well.
  • the resulting CTL or HTL cells can be used to treat chronic infections, or tumors in patients that do not respond to other conventional forms of therapy, or will not respond to a therapeutic vaccine peptide or nucleic acid in accordance with the invention.
  • Ex vivo CTL or HTL responses to a particular pathogen are induced by incubating in tissue culture the patient's CTL or HTL precursor cells together with a source of antigen-presenting cells (APC), such as dendritic cells, and the appropriate immunogenic peptide.
  • APC antigen-presenting cells
  • Transfected dendritic cells may also be used as antigen presenting cells.
  • dendritic cells are transfected, e.g., with a minigene construct in accordance with the invention, in order to elicit immune responses. Minigenes will be discussed in greater detail in a following section.
  • DNA or RNA encoding one or more of the peptides of the invention can also be administered to a patient.
  • This approach is described, for instance, in Wolff et. al., Science 247:1465 (1990) as well as U.S. Pat. Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; WO 98/04720; and in more detail below.
  • Examples of DNA-based delivery technologies include “naked DNA”, facilitated (bupivicaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) delivery.
  • the following principles are utilized when selecting an array of epitopes for inclusion in a polyepitopic composition, or for selecting epitopes to be included in a vaccine composition and/or to be encoded by a minigene. It is preferred that each of the following principles are balanced in order to make the selection.
  • Epitopes are selected which, upon administration, mimic immune responses that have been observed to be correlated with HBV clearance.
  • HLA Class I this includes 3-4 epitopes that come from at least one antigen of HBV.
  • HLA Class II a similar rationale is employed; again 3-4 epitopes are selected from at least one HBV antigen (see e.g., Rosenberg et al. Science 278:1447-1450).
  • Epitopes are selected that have the requisite binding affinity established to be correlated with immunogenicity: for HLA Class I an IC 50 of 500 nM or less, or for Class II an IC 50 of 1000 nM or less.
  • Sufficient supermotif bearing peptides, or a sufficient array of allele-specific motif bearing peptides, are selected to give broad population coverage. For example, it is preferable to have at least 80% population coverage.
  • a Monte Carlo analysis a statistical evaluation known in the art, can be employed to assess population coverage.
  • nested epitopes When selecting epitopes from cancer-related antigens it is often preferred to select analogs. When selecting epitopes for infectious disease-related antigens it is often preferable to select native epitopes. Therefore, of particular relevance for infectious disease vaccines (but for cancer-related vaccines as well), are epitopes referred to as “nested epitopes.” Nested epitopes occur where at least two epitopes overlap in a given peptide sequence.
  • a peptide comprising “transcendent nested epitopes” is a peptide that has both HLA class I and HLA class II epitopes in it.
  • a sequence that has the greatest number of epitopes per provided sequence it is preferable to provide a sequence that has the greatest number of epitopes per provided sequence.
  • a limitation on this principle is to avoid providing a peptide that is any longer than the amino terminus of the amino terminal epitope and the carboxyl terminus of the carboxyl terminal epitope in the peptide.
  • a longer peptide sequence such as a sequence comprising nested epitopes, it is important to screen the sequence in order to insure that it does not have pathological or other deleterious biological properties.
  • an objective is to generate the smallest peptide possible that encompasses the epitopes of interest.
  • the principles employed are similar, if not the same as those employed when selecting a peptide comprising nested epitopes.
  • the peptide encoded thereby is analyzed to determine whether any “junctional epitopes” have been created.
  • a junctional epitope is an actual binding epitope, as predicted, e.g., by motif analysis. Junctional epitopes are to be avoided because the recipient may generate an immune response to that epitope. Of particular concern is a junctional epitope that is a “dominant epitope.” A dominant epitope may lead to such a zealous response that immune responses to other epitopes are diminished or suppressed.
  • Nucleic acids encoding the peptides of the invention are a particularly useful embodiment of the invention. Epitopes for inclusion in a minigene are preferably selected according to the guidelines above.
  • a preferred means of administering nucleic acids encoding the peptides of the invention uses minigene constructs encoding one or multiple epitopes of the invention. The use of multi-epitope minigenes is described below and in, e.g. An, L. and Whitton, J. L., J. Virol. 71:2292, 1997; Thomson, S. A. et al., J. Immunol.
  • the amino acid sequences of the epitopes may be reverse translated.
  • a human codon usage table can be used to guide the codon choice for each amino acid.
  • These epitope-encoding DNA sequences may be directly adjoined, so that when translated, a continuous polypeptide sequence is created.
  • additional elements can be incorporated into the minigene design.
  • amino acid sequences that could be reverse translated and included in the minigene sequence include: HLA class I epitopes, HLA class II epitopes, a ubiquitination signal sequence, a leader sequence, and/or an endoplasmic reticulum targeting signal.
  • HLA presentation of CTL and HTL epitopes may be improved by including synthetic (e.g. poly-alanine) or naturally-occurring flanking sequences adjacent to the CTL or HTL epitopes.
  • the minigene sequence may be converted to DNA by assembling oligonucleotides that encode the plus and minus strands of the minigene. Overlapping oligonucleotides (30-100 bases long) may be synthesized, phosphorylated, purified and annealed under appropriate conditions using well known techniques. The ends of the oligonucleotides can be joined, for example, using T4 DNA ligase. This synthetic minigene, encoding the epitope polypeptide, can then be cloned into a desired expression vector.
  • Standard regulatory sequences well known to those of skill in the art are preferably included in the vector to ensure expression in the target cells.
  • a promoter with a down-stream cloning site for minigene insertion a polyadenylation signal for efficient transcription termination; an E. coli origin of replication; and an E. coli selectable marker (e.g. ampicillin or kanamycin resistance).
  • Numerous promoters can be used for this purpose, e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat. Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.
  • introns are required for efficient gene expression, and one or more synthetic or naturally-occurring introns could be incorporated into the transcribed region of the minigene.
  • mRNA stabilization sequences and sequences for replication in mammalian cells may also be considered for increasing minigene expression.
  • the minigene is cloned into the polylinker region downstream of the promoter.
  • This plasmid is transformed into an appropriate E. coli strain, and DNA is prepared using standard techniques. The orientation and DNA sequence of the minigene, as well as all other elements included in the vector, are confirmed using restriction mapping and DNA sequence analysis. Bacterial cells harboring the correct plasmid can be stored as a master cell bank and a working cell bank.
  • immunostimulatory sequences appear to play a role in the immunogenicity of DNA vaccines. These sequences may be included in the vector, outside the minigene coding sequence, if desired to enhance immunogenicity.
  • a bi-cistronic expression vector which allows production of both the minigene-encoded epitopes and a second protein (included to enhance or decrease immunogenicity) can be used.
  • proteins or polypeptides that could beneficially enhance the immune response if co-expressed include cytokines (e.g., IL-2, IL-12, GM-CSF), cytokine-inducing molecules (e.g., LeIF) or costimulatory molecules.
  • Helper (HTL) epitopes can be joined to intracellular targeting signals and expressed separately from expressed CTL epitopes; this allows direction of the HTL epitopes to a cell compartment different than that of the CTL epitopes.
  • immunosuppressive molecules e.g. TGF- ⁇
  • TGF- ⁇ immunosuppressive molecules
  • Therapeutic quantities of plasmid DNA can be produced for example, by fermentation in E. coli , followed by purification. Aliquots from the working cell bank are used to inoculate growth medium, and grown to saturation in shaker flasks or a bioreactor according to well known techniques. Plasmid DNA can be purified using standard bioseparation technologies such as solid phase anion-exchange resins supplied by QIAGEN, Inc. (Valencia, Calif.). If required, supercoiled DNA can be isolated from the open circular and linear forms using gel electrophoresis or other methods.
  • Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is reconstitution of lyophilized DNA in sterile phosphate-buffer saline (PBS). This approach, known as “naked DNA,” is currently being used for intramuscular (IM) administration in clinical trials. To maximize the immunotherapeutic effects of minigene DNA vaccines, an alternative method for formulating purified plasmid DNA may be desirable. A variety of methods have been described, and new techniques may become available. Cationic lipids can also be used in the formulation (see, e.g., as described by WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682 (1988); U.S. Pat. No.
  • glycolipids, fusogenic liposomes, peptides and compounds referred to collectively as protective, interactive, non-condensing could also be complexed to purified plasmid DNA to influence variables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types.
  • Target cell sensitization can be used as a functional assay for expression and HLA class I presentation of minigene-encoded CTL epitopes, respectively.
  • the plasmid DNA is introduced into a mammalian cell line that is suitable as a target for standard CTL chromium release assays.
  • the transfection method used will be dependent on the final formulation. Electroporation can be used for “naked” DNA, whereas cationic lipids allow direct in vitro transfection.
  • a plasmid expressing green fluorescent protein (GFP) can be co-transfected to allow enrichment of transfected cells using fluorescence activated cell sorting (FACS). These cells are then chromium-51 ( 51 Cr) labeled and used as target cells for epitope-specific CTL lines; cytolysis, detected by 51 Cr release, indicates production of HLA presentation of minigene-encoded CTL epitopes.
  • In vivo immunogenicity is a second approach for functional testing of minigene DNA formulations.
  • Transgenic mice expressing appropriate human HLA proteins are immunized with the DNA product.
  • the dose and route of administration are formulation dependent (e.g., IM for DNA in PBS, IP for lipid-complexed DNA).
  • Twenty-one days after immunization splenocytes are harvested and restimulated for 1 week in the presence of peptides encoding each epitope being tested.
  • assays are conducted for cytolysis of peptide-loaded, chromium-51 labeled target cells using standard techniques. Lysis of target cells sensitized by HLA loading of peptides corresponding to minigene-encoded epitopes demonstrates DNA vaccine function for in vivo induction of CTLs.
  • nucleic acids can be administered using ballistic delivery as described, for instance, in U.S. Pat. No. 5,204,253.
  • particles comprised solely of DNA are administered.
  • DNA can be adhered to particles, such as gold particles.
  • the peptides of the present invention, or analogs thereof, which have immunostimulatory activity may be modified to provide desired attributes, such as improved serum half life, or to enhance immunogenicity.
  • the ability of the peptides to induce CTL activity can be enhanced by linking the peptide to a sequence which contains at least one epitope that is capable of inducing a T helper cell response.
  • Particularly preferred immunogenic peptides/T helper conjugates are linked by a spacer molecule.
  • the spacer is typically comprised of relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions.
  • the spacers are typically selected from, e.g., Ala, Gly, or other neutral spacers of nonpolar amino acids or neutral polar amino acids. It will be understood that the optionally present spacer need not be comprised of the same residues and thus may be a hetero- or homo-oligomer. When present, the spacer will usually be at least one or two residues, more usually three to six residues.
  • the CTL peptide may be linked to the T helper peptide without a spacer.
  • the immunogenic peptide may be linked to the T helper peptide either directly or via a spacer either at the amino or carboxy terminus of the CTL peptide.
  • the amino terminus of either the immunogenic peptide or the T helper peptide may be acylated.
  • the T helper peptides used in the invention can be modified in the same manner as CTL peptides. For instance, they may be modified to include D-amino acids or be conjugated to other molecules such as lipids, proteins, sugars and the like.
  • Exemplary T helper peptides include tetanus toxoid 830-843, influenza 307-319, and malarial circumsporozoite 382-398 and 378-389.
  • the T helper peptide is one that is recognized by T helper cells present in the majority of the population. This can be accomplished by selecting amino acid sequences that bind to many, most, or all of the HLA class II molecules. These are known as “loosely HLA-restricted” or “promiscuous” T helper sequences.
  • amino acid sequences that are promiscuous include sequences from antigens such as tetanus toxoid at positions 830-843 (QYIKANSKFIGITE; SEQ ID NO:2572), Plasmodium falciparum CS protein at positions 378-398 (DIEKKIAKMEKASSVFNVVNS; SEQ ID NO:2573), and Streptococcus 18 kD protein at positions 116 (GAVDSILGGVATYGAA; SEQ ID NO:2574).
  • antigens such as tetanus toxoid at positions 830-843 (QYIKANSKFIGITE; SEQ ID NO:2572), Plasmodium falciparum CS protein at positions 378-398 (DIEKKIAKMEKASSVFNVVNS; SEQ ID NO:2573), and Streptococcus 18 kD protein at positions 116 (GAVDSILGGVATYGAA; SEQ ID NO:2574).
  • Other examples include peptides
  • pan-DR-binding epitope peptide having the formula: aKXVWANTLKAAa, where “X” is either cyclohexylalanine, phenylalanine, or tyrosine, and a is either D-alanine or L-alanine (SEQ ID NO:2575), has been found to bind to most HLA-DR alleles, and to stimulate the response of T helper lymphocytes from most individuals, regardless of their HLA type.
  • T helper epitopes can also be modified to alter their biological properties.
  • peptides presenting T helper epitopes can contain D-amino acids to increase their resistance to proteases and thus extend their serum half-life.
  • the epitope peptides of the invention can be conjugated to other molecules such as lipids, proteins or sugars, or any other synthetic compounds, to increase their biological activity.
  • the T helper peptide can be conjugated to one or more palmitic acid chains at either the amino or carboxyl termini.
  • compositions of the invention at least one component which primes cytotoxic T lymphocytes.
  • Lipids have been identified as agents capable of priming CTL in vivo against viral antigens.
  • palmitic acid residues can be attached to the ⁇ - and ⁇ -amino groups of a lysine residue and then linked, e.g., via one or more linking residues such as Gly, Gly-Gly-, Ser, Ser-Ser, or the like, to an immunogenic peptide.
  • lipidated peptide can then be administered either directly in a micelle or particle, incorporated into a liposome, or emulsified in an adjuvant, e.g., incomplete Freund's adjuvant.
  • a particularly effective immunogenic comprises palmitic acid attached to ⁇ - and ⁇ -amino groups of Lys, which is attached via linkage, e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.
  • E. coli lipoproteins such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P 3 CSS) can be used to prime virus specific CTL when covalently attached to an appropriate peptide.
  • P 3 CSS tripalmitoyl-S-glycerylcysteinlyseryl-serine
  • P 3 CSS tripalmitoyl-S-glycerylcysteinlyseryl-serine
  • amino acids can be added to the termini of a peptide to provide for ease of linking peptides one to another, for coupling to a carrier support, or larger peptide, for modifying the physical or chemical properties of the peptide or oligopeptide, or the like.
  • Amino acids such as tyrosine, cysteine, lysine, glutamic or aspartic acid, or the like, can be introduced at the C- or N-terminus of the peptide or oligopeptide, particularly class I peptides.
  • modification at the carboxyl terminus may, in some cases, alter binding characteristics of the peptide.
  • the peptide or oligopeptide sequences can differ from the natural sequence by being modified by terminal-NH 2 acylation, e.g., by alkanoyl (C 1 -C 20 ) or thioglycolyl acetylation, terminal-carboxyl amidation, e.g., ammonia, methylamine, etc. In some instances these modifications may provide sites for linking to a support or other molecule.
  • compositions containing the peptides of the invention are administered to a patient susceptible to or otherwise at risk for HBV infection to elicit an immune response against HBV antigens and thus enhance the patient's own immune response capabilities.
  • compositions are administered to a patient in an amount sufficient to elicit an effective CTL response to the virus or tumor antigen and to cure or at least partially arrest or slow symptoms and/or complications.
  • Amounts effective for this use will depend on, e.g., the particular composition administered, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician.
  • the dosage range for an initial immunization is between about 1.0 ⁇ g to about 5000 ⁇ g of peptide, typically between about 10 ⁇ g to about 1000 mg, for a 70 kg patient, followed by boosting dosages of between about 1.0 ⁇ g to about 5000 ⁇ g of peptide pursuant to a boosting regimen over weeks to months depending upon the patient's response and condition as determined by measuring specific CTL activity in the patient's blood.
  • the peptides and compositions of the present invention may be employed in serious disease states, that is, life-threatening or potentially life threatening situations.
  • the “CTL” peptides of the invention induce immune responses when contacted with a CTL specific to an epitope comprised by the peptide.
  • the manner in which the peptide is contacted with the CTL is not critical to the invention.
  • the peptide can be contacted with the CTL either in vivo or in vitro. If the contacting occurs in vivo, the peptide itself can be administered to the patient, or other vehicles, e.g., DNA vectors encoding one or more peptides, viral vectors encoding the peptide(s), liposomes and the like, can be used, as described herein.
  • the immunogenic peptides, or DNA encoding them are generally administered to an individual already infected with HBV.
  • the peptides or DNA encoding them can be administered individually or as fusions of one or more peptide sequences.
  • Those in the incubation phase or the acute phase of infection can be treated with the immunogenic peptides separately or in conjunction with other treatments, as appropriate.
  • administration should generally begin at the first diagnosis of HBV infection. This is followed by boosting doses until at least symptoms are substantially abated and for a period thereafter. In chronic infection, loading doses followed by boosting doses may be required.
  • compositions of the invention may hasten resolution of the infection in acutely infected individuals.
  • the compositions are particularly useful in methods for preventing the evolution from acute to chronic infection. Where susceptible individuals are identified prior to or during infection, the composition can be targeted to them, minimizing need for administration to a larger population.
  • a representative dose is in the range of about 1.0 ⁇ g to about 5000 ⁇ g, preferably about 10 ⁇ g to 1000 ⁇ g, per 70 kg patient weight per dose.
  • administration should continue until at least clinical symptoms or laboratory tests indicate that the viral infection has been eliminated or substantially abated and for a period thereafter.
  • the dosages, routes of administration, and dose schedules are adjusted in accordance with methodologies known in the art.
  • compositions for therapeutic treatment are intended for parenteral, topical, oral, intrathecal, or local administration.
  • the pharmaceutical compositions are administered parentally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly.
  • the invention provides compositions for parenteral administration which comprise a solution of the immunogenic peptides dissolved or suspended in an acceptable carrier, preferably an aqueous carrier.
  • an acceptable carrier preferably an aqueous carrier.
  • aqueous carriers may be used, e.g., water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like.
  • These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered.
  • compositions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • concentration of peptides of the invention in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
  • the peptides of the invention may also be administered via liposomes, which serve to target the peptides to a particular tissue, such as lymphoid tissue, or targeted selectively to infected cells, as well as increase the half-life of the peptide composition.
  • Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like.
  • the peptide to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to, e.g., a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions.
  • liposomes either filled or decorated with a desired peptide of the invention can be directed to the site of lymphoid cells, where the liposomes then deliver the peptide compositions.
  • Liposomes for use in the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka, et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.
  • a ligand to be incorporated into the liposome can include, e.g., antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells.
  • a liposome suspension containing a peptide may be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the manner of administration, the peptide being delivered, and the stage of the disease being treated.
  • nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more peptides of the invention, and more preferably at a concentration of 25%-75%.
  • the immunogenic peptides are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of peptides are 0.01%-20% by weight, preferably 1%-10%.
  • the surfactant must, of course, be nontoxic, and preferably soluble in the propellant.
  • Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride.
  • Mixed esters such as mixed or natural glycerides may be employed.
  • the surfactant may constitute 0.1%-20% by weight of the composition, preferably 0.25-5%.
  • the balance of the composition is ordinarily propellant.
  • a carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery.
  • the vaccine compositions of the invention may also be used purely as prophylactic agents.
  • Vaccine compositions containing the peptide epitopes of the invention are administered to a patient susceptible to, or otherwise at risk for, HBV infection to elicit an immune response against HBV antigens and thus enhance the patient's own immune response capabilities following exposure to HBV.
  • the dosage range for an initial prophylactic immunization is between about 1.0 ⁇ g to about 5000 ⁇ g of peptide, typically between about 10 ⁇ g to about 1000 ⁇ g, for a 70 kg patient.
  • This is followed by boosting dosages of between about 1.0 ⁇ g to about 5000 ⁇ g of peptide administered at defined intervals from about four weeks to six months after the initial administration of vaccine.
  • the immunogenicity of the vaccine may be assessed by measuring specific CTL activity in the patient's blood.
  • kits can be provided in kit form together with instructions for vaccine administration.
  • the kit would include desired peptide compositions in a container, preferably in unit dosage form and instructions for administration.
  • An alternative kit would include a minigene construct with desired nucleic acids of the invention in a container, preferably in unit dosage form together with instruction for administration. Lymphokines such as IL-2 or IL-12 may also be included in the kit.
  • kit components that may also be desirable include, for example, a sterile syringe, booster dosages, and other desired excipients.
  • peptide binding to HLA-A3 supertype molecules demonstrates quantification of binding affinities of HLA class I peptides.
  • Analogous binding assays can be performed for other peptides that bind class I or class II HLA molecules.
  • binding assays can be performed with peptides that are not motif-bearing.
  • Epstein-Barr virus (EBV)-transformed homozygous cell lines were used as sources of class I molecules.
  • Cell lines include, e.g., GM3107 (A3, B7; Human Genetic Mutant Repository); BVR (A11, B35.3, Cw4; Human Genetic Mutant Repository); SPACH (A31, B62, Cw1/3; ASHI Repository Collection); LWAGS (A*3301, B14, and Cw8; ASHI Repository Collection) (Bodmer, et al., Hum. Immunol. 43:149, 1995), and a C1R transfectant characterized by Dr.
  • ESV Epstein-Barr virus
  • Cell lysates were prepared and HLA class I molecules purified in accordance with disclosed protocols (Sidney, et al., J. Immunol. 154:247 (1995); Sette, et al., Mol. Immunol. 31:813 (1994)). Briefly, cells were lysed at a concentration of 10 8 cells/ml in 50 mM Tris-HCl, pH 8.5, containing 1% Nonidet P-40 (Fluka Biochemika, Buchs, Switzerland), 150 mM NaCl, 5 mM EDTA, and 2 mM PMSF. The lysates were passed through 0.45 ⁇ M filters and cleared of nuclei and debris by centrifugation at 10,000 g for 20 minutes.
  • HLA proteins were then purified by affinity chromatography. Columns of inactivated Sepharose CL 4B and Protein A Sepharose were used as precolumns. The cell lysate was depleted of HLA-B and HLA-C proteins by repeated passage over Protein A Sepharose beads conjugated with the anti-HLA(B,C) antibody B1.23.2 (Rebai, et al., Tissue Antigens 22:107 (1983)). Typically two to four passages were required for effective depletion. Subsequently, the anti HLA(A,B,C) antibody W6/32 (Barnstable, et al., Cell 14:9 (1978)) was used to capture HLA-A molecules. Protein purity, concentration, and effectiveness of depletion steps were monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • Quantitative assays for the binding of peptides to soluble class I molecules on the basis of the inhibition of binding of a radiolabeled standard probe peptide to detergent solubilized HLA molecules were performed as described in the literature (Kubo, et al., J. Immunol. 152:3913 (1994); Kast, et al., J. Immunol. 152:3904 (1994); Sidney, et al., J. Immunol. 154:247 (1995); Sette, et al., Mol. Immunol. 31:813 (1994); Ruppert, et al., Cell 74:929 (1993)).
  • the A3CON1 peptide (sequence KVFPYALINK; SEQ ID NO:2576) (Kubo, et al., J. Immunol. 152:3913 (1994)) was used as the radiolabeled probe for the A3, A11, A31, and A*6801 assays.
  • a T7Y analogue of HBVc 141-151 (sequence STLPETYVVRR; SEQ ID NO:2577) (Missale, et al., J. Exp. Med. 177:751 (1993) was used as the radiolabeled probe for the A*3301 assay.
  • the concentration of peptide yielding 50% inhibition of the binding of the radiolabeled probe peptide (IC 50 ) was calculated. Peptides were usually tested at one or two high doses, and the IC 50 of peptides yielding positive inhibition were determined in subsequent experiments, in which two to six further dilutions were tested, as necessary. To achieve a suitable signal, HLA concentrations yielding approximately 15% binding of the radiolabeled probe peptide were used for all competitive inhibition assays. Under these conditions the concentration of the labeled peptide is less than the concentration of the HLA molecule and the IC 50 is less than the concentration of the HLA molecule, therefore the measured IC 50 s are reasonable approximations of the true K D values.
  • Each competitor peptide was tested in two to four completely independent experiments. As a positive control, in each experiment, the unlabeled version of the relevant radiolabeled probe was tested and its IC 50 measured.
  • the average IC 50 of A3CON1 for the A3, A11, A31, and A*6801 assays were 11, 6, 18, and 8 nM, respectively.
  • the average IC 50 of the HBVc 141-151 peptide in the A*3301 assay was 29 nM.
  • HLA motifs and supermotifs are useful in preparing highly cross-reactive native peptides, as demonstrated herein.
  • definition of HLA motifs and supermotifs also allows one to engineer highly cross-reactive epitopes by identifying residues within a native peptide sequence which can be analoged, or “fixed”, to confer upon a peptide certain characteristics, e.g., greater cross-reactivity within the group of HLA molecules that make-up the supertype, and/or greater binding affinity for some or all of those HLA molecules Examples of analog peptides that exhibit modulated binding affinity are provided.
  • HLA supermotifs are of value in engineering highly cross-reactive peptides by identifying particular residues at secondary anchor positions that are associated with such cross-reactive properties. Demonstrating this, the capacity of a second set of peptides representing discreet single amino acid substitutions at positions one and three of five different B7-supertype binding peptides were synthesized and tested for their B-7 supertype binding capacity. In 4/4 cases the effect of replacing the native residue at position 1 with the aromatic residue F (an “F1” substitution) resulted in an increase in cross-reactivity, compared to the parent peptide, and, in most instances, binding affinity was increased three-fold or better (Table XXII).
  • HBV env 313, MAGE2 170, and HCV core 168 complete supertype cross-reactivity was achieved with the F1 substitution analogs. These gains were achieved by dramatically increasing B*5401 binding affinity. Also, gains in affinity were noted for other alleles in the cases of HCV core 168 (B*3501 and B*5301) and MAGE2 170 (B*3501, B51 and B*5301). Finally, in the case of MAGE3 196, the F1 replacement was effective in increasing cross-reactivity because of gains in B*0702 binding. An almost 70-fold increase in B51 binding capacity was also noted.
  • HLA class I binding peptides can be assessed in vivo as described in, e.g., Sette et al. J. Immunol. 153:5586-5592 (1994).
  • This example illustrates such a procedure, whereby subcutaneous injection of HBV peptide in Incomplete Freund's Adjuvant (IFA) can be used to induce HBV-specific CTL in mice that are transgenic for a human HLA allele such as the human HLA-A11 allele.
  • IFA Incomplete Freund's Adjuvant
  • mice that are transgenic for HLA-A11 are injected with 100 microliters of an emulsion of purified HBV peptide in IFA.
  • the purified peptide comprises an A11 motif, and is selected from the preferred peptides listed in Table XVI or, alternatively, may be an analog peptide.
  • the peptide epitope (50 ⁇ g/mouse) and equimolar amounts of the helper epitope HBV core 128-140 (140 ⁇ g/mouse) are dissolved in PBS/5% DMSO, emulsified in WA, and injected subcutaneously at the base of the tail of the transgenic mice. Eleven days after priming, splenocytes (5 ⁇ 10 6 cells/well in a 24-well plate) obtained from these animals are restimulated with syngeneic irradiated LPS blasts (2 ⁇ 10 6 /well) coated with peptide.
  • LPS blasts from unprimed HLA-A11 transgenic mice are prepared 72 hours before use by suspending splenocytes in medium containing LPS (25 ⁇ g/ml) and dextran sulfate (7 ⁇ g/ml). Coating is achieved by incubating 50 ⁇ g of peptide with 1.2 ⁇ 10 6 LPS blasts in a volume of 0.4 ml of RPMI medium supplemented with 10% FCS for 1 hour at 37° C. The cells are washed once and then co-cultured with splenocytes. After six days, effector cells are assayed, as outlined for example in Example 5, for cytotoxicity against 51 Cr-labeled 3A4-721.221-A11/K b target cells in the presence of the peptide.
  • the effector cells (2 ⁇ 10 6 cells/well) are re-stimulated at weekly intervals.
  • peptide-coated LPS blasts are used, followed by peptide-coated A11/K b cells.
  • effector cells are assayed for cytotoxicity as above.
  • This example determines that CTL induced by in vivo priming with peptide (as disclosed in Example 3) recognize endogenously synthesized antigens.
  • Effector cells from the procedure disclosed in Example 3 are re-stimulated in vitro using peptide-coated stimulator cells. Six days later, effector cells are assayed for cytotoxicity and the cell lines that contain peptide-specific cytotoxic activity are further re-stimulated. An additional six days later, these cell lines are tested for cytotoxic activity on 51 Cr labeled 3A4-721.221-A11/K b target cells, in the absence or presence of peptide, and also tested on 51 Cr labeled target cells bearing the endogenously synthesized antigen.
  • the peptide composition can comprise multiple CTL and/or HTL epitopes.
  • Such a peptide composition can comprise a lipidated HTL epitope conjugated to a preferred CTL epitope containing, for example, an A11 motif or an analog of that epitope.
  • Lipopeptides are prepared by coupling the appropriate fatty acid to the amino terminus of the resin bound peptide.
  • a typical procedure is as follows: A dichloromethane solution of a four-fold excess of a pre-formed symmetrical anhydride of the appropriate fatty acid is added to the resin and the mixture is allowed to react for two hours. The resin is washed with dichloromethane and dried. The resin is then treated with trifluoroacetic acid in the presence of appropriate scavengers [e.g. 5% (v/v) water] for 60 minutes at 20° C. After evaporation of excess trifluoroacetic acid, the crude peptide is washed with diethyl ether, dissolved in methanol and precipitated by the addition of water. The peptide is collected by filtration and dried.
  • appropriate scavengers e.g. 5% (v/v) water
  • Peptide Compositions are Typically resuspended in DMSO at a concentration of 20 mg/ml. Before use, peptides are prepared at the required concentration by dilution in saline or the appropriate medium.
  • mice which are transgenic for the human HLA A11 allele, are primed subcutaneously (base of the tail) with 0.1 ml of peptide conjugate formulated in saline, or DMSO/saline. Seven days after priming, splenocytes obtained from these animals are restimulated with syngeneic irradiated LPS-activated lymphoblasts coated with peptide.
  • RPMI-1640 supplemented with 10% fetal calf serum (FCS) 2 mM Glutamine, 50 ⁇ g/ml Gentamicin and 5 ⁇ 10 ⁇ 5 M 2-mercaptoethanol serves as culture medium
  • RPMI-1640 containing 25 mM HEPES buffer and supplemented with 2% (FCS) is used as cell washing medium.
  • the 3A4-721.221-A11/K b cell line is used as target cells.
  • This cell line is an EBV transformed cell line that was mutagenized and selected to be Class I negative which was transfected with an HLA-A11/K b gene.
  • LPS-activated lymphoblasts Splenocytes obtained from transgenic mice are resuspended at a concentration of 1-1.5 ⁇ 10 6 /m1 in culture medium supplemented with 25 ⁇ g/ml LPS and 7 ⁇ g/ml dextran sulfate in 75 cm 2 tissue culture flasks. After 72 hours at 37° C., the lymphoblasts are collected for use by centrifugation.
  • Peptide coating of lymphoblasts is achieved by incubating 30 ⁇ 10 6 irradiated (3000 rads) lymphoblasts with 100 ⁇ g of peptide in 1 ml of R10 medium for 1 hr at 37° C. Cells are then washed once and resuspended in culture medium at the desired concentration.
  • spleen cells (30 ⁇ 10 6 cells/flask) are co-cultured at 37° C. with syngeneic, irradiated (3000 rads), peptide coated lymphoblasts (10 ⁇ 10 6 cells/flask) in 10 ml of culture medium/T25 flask. After six days, the effector cells are harvested and assayed for cytotoxic activity.
  • Target cells (1.0-1.5 ⁇ 10 6 ) are incubated at 37° C. in the presence of 200 ⁇ l of sodium 51 Cr chromate. After 60 minutes, cells are washed three times and resuspended in R10 medium. Peptide is added where required at a concentration of 1 ⁇ g/ml.
  • 104 51 Cr-labeled target cells are added to different concentrations of effector cells (final volume of 200 ⁇ l) in U-bottom 96-well plates. After a 6 hour incubation period at 37° C., a 0.1 ml aliquot of supernatant is removed from each well and radioactivity is determined in a Micromedic automatic gamma counter.
  • % 51 Cr release data is expressed as lytic units/10 6 cells.
  • One lytic unit is arbitrarily defined as the number of effector cells required to achieve 30% lysis of 10,000 target cells in a 6 hour 51 Cr release assay.
  • the lytic units/10 6 obtained in the absence of peptide is subtracted from the lytic units/10 6 obtained in the presence of peptide.
  • the results are analyzed to assess the magnitude of the CTL responses of animals injected with the immunogenic CTL/HTL conjugate vaccine preparation. Analyses similar to this may be performed to evaluate the immunogenicity of peptide conjugates containing multiple CTL epitopes and/or multiple HTL epitopes. In accordance with these procedures it is found that CTL and HTL responses are induced.
  • a human clinical trial for an immunogenic composition comprising CTL and HTL epitopes is set up as an IND Phase I, dose escalation study (5, 50 and 500 ⁇ g) and carried out as a randomized, double-blind, placebo-controlled trial.
  • Such a trial is designed, for example, as follows:
  • a total of about 27 subjects are enrolled and divided into 3 groups:
  • Group I 3 subjects are injected with placebo and 6 subjects are injected with 5 ⁇ g of peptide composition
  • Group II 3 subjects are injected with placebo and 6 subjects are injected with 50 ⁇ g peptide composition;
  • Group III 3 subjects are injected with placebo and 6 subjects are injected with 500 ⁇ g of peptide composition.
  • the endpoints measured in this study relate to the safety and tolerability of the peptide composition as well as its immunogenicity.
  • Cellular immune responses to the peptide composition are an index of the intrinsic activity of this the peptide composition, and can therefore be viewed as a measure of biological efficacy.
  • Peripheral blood mononuclear cells are isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation, aliquoted in freezing media and stored frozen. Samples are assayed for CTL and HTL activity.
  • the vaccine is found to be both safe and efficacious.
  • Phase II trials are performed to study the effect of administering the CTL-HTL peptide compositions to patients (male and female) having chronic HBV infection.
  • a main objective of the trials is to determine an effective dose and regimen for inducing CTLs in chronically infected HBV patients, to establish the safety of inducing a CTL response in these patients, and to see to what extent activation of CTLs improves the clinical picture of chronically infected CTL patients, as manifested by a transient flare in alanine aminotransferase (ALT), normalization of ALT, and reduction in HBV DNA.
  • ALT alanine aminotransferase
  • Such a study is designed, for example, as follows:
  • the studies are performed in multiple centers in the U.S. and Canada.
  • the trial design is an open-label, uncontrolled, dose escalation protocol wherein the peptide composition is administered as a single dose followed six weeks later by a single booster shot of the same dose.
  • the dosages are 50, 500 and 5,000 micrograms per injection. Drug-associated adverse effects are recorded.
  • the first group is injected with 50 micrograms of the peptide composition and the second and third groups with 500 and 5,000 micrograms of peptide composition, respectively.
  • the patients within each group range in age from 21-65 and include both males and females.
  • the patients represent diverse ethnic backgrounds. All of them are infected with HBV for over five years and are HIV, HCV and HDV negative, but have positive levels of HBe antigen and HBs antigen.
  • the magnitude and incidence of ALT flares and the levels of HBV DNA in the blood are monitored to assess the effects of administering the peptide compositions.
  • the levels of HBV DNA in the blood are an indirect indication of the progress of treatment.
  • the vaccine composition is found to be both safe and efficacious in the treatment of chronic HBV infection.
  • This example illustrates the procedure for the selection of peptide epitopes for vaccine compositions of the invention.
  • the following principles are utilized when selecting an array of epitopes for inclusion in a polyepitopic composition, or for selecting epitopes to be included in a vaccine composition and/or to be encoded by a minigene. Each of the following principles are balanced in order to make the selection.
  • Epitopes are selected which, upon administration, mimic immune responses that have been observed to be correlated with HBV clearance.
  • HLA Class I this includes 3-4 epitopes that come from at least one antigen of HBV.
  • HLA Class II a similar rationale is employed; again 3-4 epitopes are selected from at least one HBV antigen.
  • Epitopes are selected that have the requisite binding affinity established to be correlated with immunogenicity: for HLA Class I an IC 50 of 500 nM or less, or for Class II an IC 50 of 1000 nM or less.
  • Sufficient supermotif bearing peptides, or a sufficient array of allele-specific motif bearing peptides, are selected to give broad population coverage.
  • epitopes are selected to provide at least 80% population coverage.
  • a Monte Carlo analysis a statistical evaluation known in the art, is employed to assess population coverage.
  • nested epitopes When selecting epitopes for HBV antigens it is often preferable to select native epitopes. Therefore, of particular relevance for infectious disease vaccines, are epitopes referred to as “nested epitopes.” Nested epitopes occur where at least two epitopes overlap in a given peptide sequence. A peptide comprising “transcendent nested epitopes” is a peptide that has both HLA class I and HLA class II epitopes in it.
  • a sequence that has the greatest number of epitopes per provided sequence is provided.
  • a limitation on this principle is to avoid providing a peptide that is any longer than the amino terminus of the amino terminal epitope and the carboxyl terminus of the carboxyl terminal epitope in the peptide.
  • the sequence is screened in order to insure that it does not have pathological or other deleterious biological properties.
  • an objective is to generate the smallest peptide possible that encompasses the epitopes of interest.
  • the principles employed are similar, if not the same as those employed when selecting a peptide comprising nested epitopes.
  • the peptide encoded thereby is analyzed to determine whether any “junctional epitopes” have been created.
  • a junctional epitope is an actual binding epitope, as predicted, e.g., by motif analysis. Junctional epitopes are to be avoided because the recipient may generate an immune response to that epitope. Of particular concern is a junctional epitope that is a “dominant epitope.” A dominant epitope may lead to such a zealous response that immune responses to other epitopes are diminished or suppressed.
  • Peptide epitopes for inclusion in vaccine compositions are, for example, selected from those listed in Table XXIII.
  • a vaccine composition comprised of selected peptides, when administered, is safe, efficacious, and elicits an immune response similar in magnitude of an immune response that clears an acute HBV infection.
  • Expression plasmids have been constructed and evaluated as described, for example, in U.S. Ser. No. 60/085,751 filed May 15, 1998 and U.S. Ser. No. 09/078,904 filed May 13, 1998.
  • the binding peptide epitopes and their positions for some of the plasmids described therein are shown in FIG. 1 as example of the orientation of peptide epitopes in minigene constructs.
  • Such a plasmid may, for example, also include multiple CTL and HTL peptide epitopes.
  • HLA-A11 motif-bearing peptides are used in conjunction with DR supermotif-bearing peptides.
  • A11 epitopes are identified, for example, in Table XVI or Table XXI and peptide epitopes recognized by HLA DR molecules (Tables XVIII and XIX).
  • Tables XVIIII and XIX Four class I A11 motif-bearing peptide epitopes or analogs of those peptide epitopes derived from the same HBV antigen, e.g. the envelope protein, are selected as CTL epitopes.
  • CTL epitopes Four class II motif-bearing peptide epitopes derived from the same antigen, e.g., the envelope protein, are selected as HTL epitopes. These epitopes are then incorporated into a minigene for expression in an expression vector.
  • This example illustrates the methods to be used for construction of such a minigene-bearing expression plasmid.
  • Other expression vectors that may be used for minigene compositions are available and known to those of skill in the art.
  • a pMin minigene DNA plasmid is constructed from an early generation DNA plasmid designated as pMin.0.
  • This plasmid contains a consensus Kozak sequence and a consensus murine kappa Ig-light chain signal sequence followed by a string of CTL and HTL epitopes selected in accordance with principles disclosed herein.
  • the pMIN sequence encodes an open reading frame fused to the Myc and His antibody epitope tag coded for by the pcDNA 3.1 Myc-His vector.
  • Overlapping oligonucleotides for example eight oligonucleotides, averaging approximately 70 nucleotides in length with 15 nucleotide overlaps, are synthesized and HPLC-purified.
  • the oligonucleotides encode the selected peptide epitopes as well as appropriate linker nucleotides.
  • the final multiepitope minigene is assembled by extending the overlapping oligonucleotides in three sets of reactions using PCR.
  • a Perkin/Elmer 9600 PCR machine is used and a total of 30 cycles are performed using the following conditions: 95° C. for 15 sec, annealing temperature (5° below the lowest calculated Tm of each primer pair) for 30 sec, and 72° C. for 1 min.
  • the full-length dimer products are gel-purified, and two reactions containing the product of 1+2 and 3+4, and the product of 5+6 and 7+8 are mixed, annealed, and extended for 10 cycles. Half of the two reactions are then mixed, and 5 cycles of annealing and extension carried out before flanking primers are added to amplify the full length product for 25 additional cycles.
  • the full-length product is gel-purified and cloned into pCR-blunt (Invitrogen) and individual clones are screened by sequencing.
  • HLA-A11/K b transgenic mice are immunized intramuscularly with 100 ⁇ g of naked cDNA.
  • a control group of animals is also immunized with an actual peptide composition that comprises multiple epitopes synthesized as a single polypeptide.
  • Splenocytes from immunized animals are stimulated twice with each of the peptide epitopes encoded in the minigene, then assayed for peptide-specific cytotoxic activity in a 51 Cr release assay.
  • the results indicate the magnitude of the CTL response directed against each of its A11-restricted epitopes, thus indicating the in vivo immunogenicity of the minigene vaccine. It is, therefore, found that the minigene elicits immune responses directed toward A11-restricted epitopes.
  • Vaccine compositions of the present invention are used to prevent HBV infection in persons who are at risk.
  • a polyepitopic peptide epitope composition containing multiple CTL and HTL epitopes such as those selected in Examples 9 and/or 10, which are also selected to target greater than 80% of the population is administered to individuals at risk for HBV infection.
  • the composition is provided as a single lipidated polypeptide that encompasses multiple epitopes.
  • the vaccine is administered in an aqueous carrier comprised of Freunds Incomplete Adjuvant.
  • the dose of peptide for the initial immunization is from about 1 to about 5,000 ⁇ g for a 70 kg patient.
  • the initial administration of vaccine is followed by booster dosages at 4 weeks followed by evaluation of the magnitude of the immune response in the patient by techniques that determine the presence of epitope-specific CTL populations in a PBMC sample. Additional booster doses are administered as required.
  • the composition is found to be both safe and efficacious as a prophylaxis against HBV infection.
  • polyepitopic peptide composition can be administered as a nucleic acid in accordance with methodologies known in the art and disclosed herein.
  • a native HBV polyprotein sequence is screened, preferably using computer algorithms defined for each class I and/or class II supermotif or motif, to identify “relatively short” regions of the polyprotein that comprise multiple epitopes.
  • This relatively short sequence that contains multiple distinct, even overlapping, epitopes is selected and used to generate a minigene construct.
  • the construct is engineered to express the peptide, which corresponds to the native protein sequence.
  • the “relatively short” peptide is less than 100 amino acids in length, preferably less than 75 amino acids in length, and more preferably less than 50 amino acids in length.
  • the protein sequence of the vaccine composition is selected because it has maximal number of epitopes contained within the sequence.
  • epitope motifs may be overlapping (i.e., frame shifted relative to one another) with frame shifted overlapping epitopes, e.g. two 9-mer epitopes can be present in a 10 amino acid peptide.
  • Such a vaccine composition is administered for therapeutic or prophylactic purposes.
  • the vaccine composition will preferably include, for example, three CTL epitopes and at least one HTL epitope from the source antigen. Junctional sequences will be analyzed to avoid sequences containing a potentially immunodominant epitope.
  • This polyepitopic native sequence is administered either as a peptide or as a nucleic acid sequence which encodes the peptide. Alternatively, an analog can be made of this native sequence.
  • the embodiment of this example provides for the possibility that an as yet undiscovered aspect of immune system processing will apply to the native nested sequence and thereby facilitate the production of therapeutic or prophylactic immune response-inducing vaccine compositions. Additionally such an embodiment provides for the possibility of motif-bearing epitopes for an HLA makeup that is presently unknown. Furthermore, this embodiment directs the immune response to sequences that are present in native HBV antigens. Lastly, the embodiment provides an economy of scale when producing nucleic acid vaccine compositions.
  • computer programs can be derived which identify, in a target sequence, the greatest number of epitopes per sequence length.
  • HBV peptide epitopes of the present invention are used in conjunction with peptide epitopes from target antigens related to one or more other diseases, to create a vaccine composition that is useful for the prevention or treatment of HBV as well as another disease.
  • other diseases include, but are not limited to, HIV, HCV, and HPV.
  • a polyepitopic peptide composition comprising multiple CTL and HTL epitopes that target greater than 98% of the population may be created for administration to individuals at risk for both HBV and HIV infection.
  • the composition can be provided as a single polypeptide that incorporates the multiple epitopes from the various disease-associated sources.
  • Peptides of the invention may be used to analyze an immune response for the presence of specific CTL populations corresponding to HBV. Such an analysis may be performed as described by Ogg et al., Science 279:2103-2106, 1998.
  • peptides in accordance with the invention are used as a reagent for diagnostic or prognostic purposes, not as an immunogen.
  • tetramers highly sensitive human leukocyte antigen tetrameric complexes
  • tetramers may be used for a cross-sectional analysis of, for example, HBV Env-specific CTL frequencies from untreated HLA A*0201-positive individuals at different stages of infection using an HBV Env peptide containing an A2.1 extended motif.
  • Tetrameric complexes are synthesized as described (Musey et al., N. Engl. J. Med. 337:1267, 1997). Briefly, purified HLA heavy chain (A2.1 in this example) and ⁇ 2-microglobulin are synthesized by means of a prokaryotic expression system.
  • the heavy chain is modified by deletion of the transmembrane-cytosolic tail and COOH-terminal addition of a sequence containing a BirA enzymatic biotinylation site.
  • the heavy chain, ⁇ 2-microglobulin, and peptide are refolded by dilution.
  • the 45-kD refolded product isolated by fast protein liquid chromatography and then biotinylated by BirA in the presence of biotin (Sigma, St. Louis, Mo.), adenosine 5′ triphosphate and magnesium.
  • Streptavidin-phycoerythrin conjugate is added in a 1:4 molar ratio, and the tetrameric product is concentrated to 1 mg/ml. The resulting product is referred to as tetramer-phycoerythrin.
  • PBMCs Approximately one million PBMCs are centrifuged at 300 g for 5 minutes and resuspended in 50 ul of cold phosphate-buffered saline. Tri-color analysis is performed with the tetramer-phycoerythrin, along with anti-CD8-Tricolor, and anti-CD38. The PBMCs are incubated with tetramer and antibodies on ice for 30 to 60 min and then washed twice before formaldehyde fixation. Gates are applied to contain >99.98% of control samples. Controls for the tetramers include both A*0201-negative individuals and A*0201-positive uninfected donors. The percentage of cells stained with the tetramer is then determined by flow cytometry. The results indicate the number of cells in the PBMC sample that contain epitope-restricted CTLs, thereby readily indicating the stage of infection with HBV or the status of exposure to HBV or to a vaccine that elicits a protective response
  • the peptide epitopes of the invention are used as reagents to evaluate T cell responses such as acute or recall responses, in patients. Such an analysis may be performed on patients who have recovered from infection or who are chronically infected with HBV or who have been vaccinated with an HBV vaccine.
  • the class I restricted CTL response of persons at risk for HBV infection who have been vaccinated may be analyzed.
  • the vaccine may be any BBV vaccine.
  • PBMC are collected from vaccinated individuals and HLA typed.
  • Appropriate peptide reagents that, are highly conserved and, optimally, bear supermotifs to provide cross-reactivity with multiple HLA supertype family members are then used for analysis of samples derived from individuals who bear that HLA type.
  • PBMC from vaccinated individuals are separated on Ficoll-Histopaque density gradients (Sigma Chemical Co., St. Louis, Mo.), washed three times in HBSS (GIBCO Laboratories), resuspended in RPMI-1640 (GIBCO Laboratories) supplemented with L-glutamine (2 mM), penicillin (50 U/ml), streptomycin (50 ⁇ g/m1), and Hepes (10 mM) containing 10% heat-inactivated human AB serum (complete RPMI) and plated using microculture formats. Synthetic peptide is added at 10 ⁇ g/ml to each well and recombinant HBc Ag is added at 1 ⁇ g/ml to each well as a source of T cell help during the first week of stimulation.
  • Target cell lines are autologous and allogeneic EBV-transformed B-LCL that are either purchased from the American Society for Histocompatibility and Immunogenetics (ASHI, Boston, Mass.) or established from the pool of patients as described (Guilhot, et al. J. Virol. 66:2670-2678, 1992).
  • Cytotoxicity assays are performed in the following manner.
  • Target cells consist of either allogeneic HLA-matched or autologous EBV-transformed B lymphoblastoid cell line that are incubated overnight with synthetic peptide at 10 ⁇ M and labeled with 100 ⁇ Ci of 51 Cr (Amersham Corp., Arlington Heights, Ill.) for 1 hour after which they are washed four times with HBSS.
  • Cytolytic activity is determined in a standard 4-h, split well 51 Cr release assay using U-bottomed 96 well plates containing 3,000 targets/well. Stimulated PBMC are tested at E/T ratios of 20-50:1 on day 14.
  • Percent cytotoxicity is determined from the formula: 100 ⁇ [(experimental release ⁇ spontaneous release)/maximum release ⁇ spontaneous release)]. Maximum release is determined by lysis of targets by detergent (2% Triton X-100®; Sigma Chemical Co., St. Louis, Mo.). Spontaneous release is ⁇ 25% of maximum release for all experiments. The results of such an analysis will indicate to what extent HLA-restricted CTL populations have been stimulated with the vaccine. Of course, this protocol can also be used to monitor prior HBV exposure.
  • MOTIFS DR4 preferred F, M, Y, L, I, M T I V, S, T, C, P, A, M, H M, H V, W L, I, M deleterious W R W, D, E DR1 preferred M, F, L, I, V, P, A, M, Q V, M, A, T, S, P, M A, V, M W, Y L, I, C, deleterious C C, H F, D C, W, D G, D, E D 2578 DR7 preferred M, F, L, I, V, M W A, I, V, M, S, A, C, M I, V W, Y T, P, L, 2579 deleterious C G, G, R, D N G DR Supermotif M, F, L, I, V, V, M, S, T, A, C, W, Y P, L, I DR3 MOTIFS motif a L, I, V, M, F,
  • Binding Standard Affinity SEQ ID Allele Nomenclature Peptide Sequence (nM) NO: DRB1*0101 DR1 515.01 PKYVKQNTLKLAT 5.0 2495 DRB1*0301 DR3 829.02 YKTIAFDEEARR 300 2496 DRB1*0401 DR4w4 515.01 PKYVKQNTLKLAT 45 2495 DRB1*0404 DR4w14 717.01 YARFQSQTTLKQKT 50 2497 DRB1*0405 DR4w15 717.01 YARFQSQTTLKQKT 38 2497 DRB1*0701 DR7 553.01 QYIKANSKFIGITE 25 2498 DRB1*0802 DR8w2 553.01 QYIKANSKFIGITE 49 2498 DRB1*0803 DR8w3 553.01 QYIKANSKFIGITE 1600 2496 DRB1*0101 DR1 515.01 PKY
  • Table XXIII lists as a matter of example one such set of epitopes.

Abstract

This invention uses our knowledge of the mechanisms by which antigen is recognized by T cells to develop epitope-based vaccines directed towards HBV. More specifically, this application communicates our discovery of pharmaceutical compositions and methods of use in the prevention and treatment of HBV infection.

Description

    CROSS-REFERENCES TO RELATED APPLICATIONS
  • This application is a divisional of U.S. application Ser. No. 10/654,601, filed Sep. 4, 2003, which is a divisional of U.S. application Ser. No. 09/239,043, filed Jan. 27, 1999, which is herein incorporated by reference; the current application is also a continuation-in-part of U.S. application Ser. No. 09/350,401, filed Jul. 8, 1999, which is a continuation-in-part of U.S. application Ser. No. 09/239,043, filed Jan. 27, 1999, now U.S. Pat. No. 6,689,363, which is herein incorporated by reference; and is a continuation-in-part of U.S. application Ser. No. 08/347,610, filed Dec. 1, 1994, abandoned, which is herein incorporated by reference; and is a continuation-in-part of U.S. application Ser. No. 08/344,824, filed Nov. 23, 1994, abandoned, which is herein incorporated by reference; and is a continuation-in-part of U.S. application Ser. No. 08/205,713, filed Mar. 4, 1994, abandoned, which is herein incorporated by reference; and is a continuation-in-part of U.S. application Ser. No. 09/189,702, filed Nov. 10, 1998, which is herein incorporated by reference; and is a continuation-in-part of U.S. application Ser. No. 08/820,360, filed Mar. 12, 1997, abandoned, which is herein incorporated by reference; and is a continuation-in-part of U.S. application Ser. No. 08/197,484, filed Feb. 16, 1994, now U.S. Pat. No. 6,419,931, which is herein incorporated by reference; which is a continuation-in-part of U.S. application Ser. No. 07/935,811, filed Aug. 26, 1992, abandoned, which is herein incorporated by reference; which is a continuation-in-part of U.S. application Ser. No. 07/874,491, filed Apr. 27, 1992, abandoned, which is herein incorporated by reference; which is a continuation-in-part of U.S. application Ser. No. 07/827,682, filed Jan. 29, 1992, abandoned, which is herein incorporated by reference; said U.S. application Ser. No. 08/347,610 (abandoned) is a continuation-in-part of U.S. application Ser. No. 08/159,339, filed Nov. 29, 1993, now U.S. Pat. No. 6,037,135, which is herein incorporated by reference; which is a continuation-in-part of U.S. application Ser. No. 08/103,396, filed Aug. 6, 1993, abandoned, which is herein incorporated by reference; which is a continuation-in-part of U.S. application Ser. No. 08/027,746, filed Mar. 5, 1993, abandoned, which is herein incorporated by reference; which is a continuation-in-part of U.S. application Ser. No. 07/926,666, filed Aug. 7, 1992, abandoned, which is herein incorporated by reference; said U.S. application Ser. No. 08/344,824 (abandoned) is a continuation-in-part of U.S. application Ser. No. 08/278,634, filed Jul. 21, 1994, abandoned, which is herein incorporated by reference; said U.S. application Ser. No. 08/205,713 (abandoned) is a continuation-in-part of U.S. application Ser. No. 08/159,184, filed Nov. 29, 1993, abandoned, which is herein incorporated by reference; which is a continuation-in-part of U.S. application Ser. No. 08/073,205, filed Jun. 4, 1993, abandoned, which is herein incorporated by reference; which is a continuation-in-part of U.S. application Ser. No. 08/027,146, filed Mar. 5, 1993, abandoned, which is herein incorporated by reference; said U.S. application Ser. No. 09/189,702 is a continuation-in-part of said U.S. application Ser. No. 08/205,713 (abandoned); said U.S. application Ser. No. 08/820,360 (abandoned) claims the benefit of U.S. Provisional Application No. 60/013,363, filed Mar. 13, 1996, abandoned, which is herein incorporated by reference; the present application is also a continuation-in-part of U.S. application Ser. No. 08/978,291, filed Nov. 25, 1997, abandoned; which is a continuation of U.S. application Ser. No. 08/461,603, filed Jun. 5, 1995, abandoned, which is herein incorporated by reference; which is a continuation of said U.S. application Ser. No. 07/935,811 (abandoned).
  • The present application is also related to abandoned U.S. Ser. No. 08/464,234, U.S. Ser. No. 08/464,496, now U.S. Pat. No. 6,322,789, abandoned U.S. Ser. No. 08/464,031, abandoned U.S. Ser. No. 08/464,433, and abandoned U.S. Ser. No. 08/461,603, which is a continuation of abandoned U.S. Ser. No. 07/935,811, which is a CIP of abandoned U.S. Ser. No. 07/874,491, which is a CIP of abandoned U.S. Ser. No. 07/827,682, which is a CIP of abandoned 07/749,568. The present application is also related to U.S. Ser. No. 09/226,775, filed Jan. 6, 1999, which is a CIP of abandoned U.S. Ser. No. 08/815,396, which is a CIP of abandoned U.S. Ser. No. 60/013,113. Furthermore, the present application is related to abandoned U.S. Ser. No. 09/017,735, which is a CIP of abandoned U.S. Ser. No. 08/589,108; abandoned U.S. Ser. No. 08/753,622, abandoned U.S. Ser. No. 08/822,382, abandoned U.S. Ser. No. 60/013,980, abandoned U.S. Ser. No. 08/454,033, abandoned U.S. Ser. No. 09/116,424, abandoned U.S. Ser. No. 08/205,713, and abandoned U.S. Ser. No. 08/349,177, which is a CIP of abandoned U.S. Ser. No. 08/159,184, which is a CIP of abandoned U.S. Ser. No. 08/073,205, which is a CIP of abandoned U.S. Ser. No. 08/027,146. The present application is also related to abandoned U.S. Ser. No. 09/017,524, abandoned U.S. Ser. No. 08/821,739, abandoned U.S. Ser. No. 60/013,833, abandoned U.S. Ser. No. 08/758,409, abandoned U.S. Ser. No. 08/589,107, abandoned U.S. Ser. No. 08/451,913, U.S. Ser. No. 08/186,266, now U.S. Pat. No. 5,662,907, abandoned U.S. Ser. No. 09/116,061, and abandoned U.S. Ser. No. 08/347,610, which is a CIP of U.S. Ser. No. 08/159,339, now U.S. Pat. No. 6,037,135, which is a CIP of abandoned U.S. Ser. No. 08/103,396, which is a CIP of abandoned U.S. Ser. No. 08/027,746, which is a CIP of abandoned U.S. Ser. No. 07/926,666. The present application is also related to abandoned U.S. Ser. No. 09/017,743, abandoned U.S. Ser. No. 08/753,615; U.S. Ser. No. abandoned 08/590,298, abandoned U.S. Ser. No. 09/115,400, and abandoned U.S. Ser. No. 08/452,843, which is a CIP of abandoned U.S. Ser. No. 08/344,824, which is a CIP of abandoned U.S. Ser. No. 08/278,634. The present application is also related to provisional U.S. Ser. No. 60/087,192 and U.S. Ser. No. 09/009,953, now U.S. Pat. No. 6,413,517, which is a CIP of abandoned U.S. Ser. No. 60/036,713 and abandoned U.S. Ser. No. 60/037,432. In addition, the present application is related to abandoned U.S. Ser. No. 09/098,584 and to U.S. Prov. Appl. No. 60/117,486, filed Jan. 27, 1999, now inactive. All of the above applications are incorporated herein by reference.
  • FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
  • This invention was funded, in part, by the United States government under grants with the National Institutes of Health. The U.S. government has certain rights in this invention.
  • REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB
  • The content of the electronically submitted sequence listing (Name: sequence listing ascii.txt, Size: 524,288 bytes; and Date of Creation: Jul. 31, 2009) filed herewith the application is incorporated by reference in its entirety.
  • INDEX I. Background of the Invention II. Summary of the Invention III. Brief Description of the Figures IV. Detailed Description of the Invention
      • A. Definitions
      • B. Stimulation of CTL and HTL responses against HBV
      • C. Immune Response Stimulating Peptides
        • 1. Binding Affinity of the Peptides for HLA Molecules
        • 2. Peptide Binding Motifs and Supermotifs
          • a) HLA-A1 supermotif
          • b) HLA-A2 supermotif
          • c) HLA-A3 supermotif
          • d) HLA-A24 supermotif
          • e) HLA-B7 supermotif
          • f) HLA-B27 supermotif
          • g) HLA-B44 supermotif
          • h) HLA-B58 supermotif
          • i) HLA-B62 supermotif
          • j) HLA-A1 motif
          • k) HLA-A3 motif
          • l) HLA-A11 motif
          • m) HLA-A24 motif
          • n) HLA-A2.1 motif
          • o) HLA-DR-1-4-7 supermotif
          • p) HLA-DR3 motifs
        • 3. Enhancing Population Coverage of the Vaccine
      • D. Immune Response Stimulating Peptide Analogs
      • E. Computer Screening of Protein Sequences from Disease-Related Antigens for Supermotif or Motif Containing Peptides
      • F. Assays to Detect T-Cell Responses
      • G. Preparation of Peptides
      • H. Use of Peptide Epitopes for Evaluating Immune Responses
      • I. Vaccine Compositions
        • 1. Minigene Vaccines
        • 2. Combinations with Helper Peptides
      • J. Administration of Vaccines for Therapeutic or Prophylactic Purposes
      • K. Kits
    V. Examples I. BACKGROUND OF THE INVENTION
  • Chronic infection by hepatitis B virus (HBV) affects at least 5% of the world's population and is a major cause of cirrhosis and hepatocellular carcinoma (Hoofnagle, J., N. Engl. J. Med. 323:337, 1990; Fields, B. and Knipe, D., In: Fields Virology 2:2137, 1990). The World Health Organization lists hepatitis B as a leading cause of death worldwide, close behind chronic pulmonary disease, and more prevalent than AIDS. Chronic HBV infection can range from an asymptomatic carrier state to continuous hepatocellular necrosis and inflammation, and can lead to hepatocellular carcinoma.
  • The immune response to HBV is believed to play an important role in controlling hepatitis B infection. A variety of humoral and cellular responses to different regions of the HBV nucleocapsid core and surface antigens have been identified. T cell mediated immunity, particularly involving class I human leukocyte antigen-restricted cytotoxic T lymphocytes (CTL), is believed to be crucial in combating established HBV infection.
  • Class I human leukocyte antigen (HLA) molecules are expressed on the surface of almost all nucleated cells. CTL recognize peptide fragments, derived from intracellular processing of various antigens, in the form of a complex with class I HLA molecules. This recognition event then results in the destruction of the cell bearing the HLA-peptide complex directly or the activation of non-destructive mechanisms e.g., the production of interferon, that inhibit viral replication.
  • Several studies have emphasized the association between self-limiting acute hepatitis and multispecific CTL responses (Penna, A. et al., J. Exp. Med. 174:1565, 1991; Nayersina, R. et al., J. Immunol. 150:4659, 1993). Spontaneous and interferon-related clearance of chronic HBV infection is also associated with the resurgence of a vigorous CTL response (Guidotti, L. G. et al., Proc. Natl. Acad. Sci. USA 91:3764, 1994). In all such cases the CTL responses are polyclonal, and specific for multiple viral proteins including the HBV envelope, core and polymerase antigens. By contrast, in patients with chronic hepatitis, the CTL activity is usually absent or weak, and antigenically restricted.
  • The crucial role of CTL in resolution of HBV infection has been further underscored by studies using HBV transgenic mice. Adoptive transfer of HBV-specific CTL into mice transgenic for the HBV genome resulted in suppression of virus replication. This effect was primarily mediated by a non-lytic, lymphokine-based mechanism (Guidotti, L. G. et al., Proc. Natl. Acad. Sci. USA 91:3764, 1994; Guidotti, L. G., Guilhot, S., and Chisari, F. V. J. Virol. 68:1265, 1994; Guidotti, L. G. et al., J. Virol. 69:6158, 1995; Gilles, P. N., Fey, G., and Chisari, F. V., J. Virol. 66:3955, 1992).
  • As is the case for HLA class I restricted responses, HLA class II restricted T cell responses are usually detected in patients with acute hepatitis, and are absent or weak in patients with chronic infection (Chisari, F. V. and Ferrari, C., Annu. Rev. Immunol. 13:29, 1995). HLA Class II responses are tied to activation of helper T cells (HTLs) Helper T lymphocytes, which recognize Class II HLA molecules, may directly contribute to the clearance of HBV infection through the secretion of cytokines which suppress viral replication (Franco, A. et al., J. Immunol. 159:2001, 1997). However, their primary role in disease resolution is believed to be mediated by inducing activation and expansion of virus-specific CTL and B cells.
  • In view of the heterogeneous immune response observed with HBV infection, induction of a multi-specific cellular immune response directed simultaneously against multiple epitopes appears to be important for the development of an efficacious vaccine against HBV. There is a need to establish vaccine embodiments that elicit immune responses that correspond to responses seen in patients that clear HBV infection. Epitope-based vaccines appear useful.
  • Upon development of appropriate technology, the use of epitope-based vaccines has several advantages over current vaccines. The epitopes for inclusion in such a vaccine are to be selected from conserved regions of viral or tumor-associated antigens, in order to reduce the likelihood of escape mutants. The advantage of an epitope-based approach over the use of whole antigens is that there is evidence that the immune response to whole antigens is directed largely toward variable regions of the antigen, allowing for immune escape due to mutations. Furthermore, immunosuppressive epitopes that may be present in whole antigens can be avoided with the use of epitope-based vaccines.
  • Additionally, with an epitope-based vaccine approach, there is an ability to combine selected epitopes (CTL and HTL) and additionally to modify the composition of the epitopes, achieving, for example, enhanced immunogenicity. Accordingly, the immune response can be modulated, as appropriate, for the target disease. Similar engineering of the response is not possible with traditional approaches.
  • Another major benefit of epitope-based immune-stimulating vaccines is their safety. The possible pathological side effects caused by infectious agents or whole protein antigens, which might have their own intrinsic biological activity, is eliminated.
  • An epitope-based vaccine also provides the ability to direct and focus an immune response to multiple selected antigens from the same pathogen. Thus, patient-by-patient variability in the immune response to a particular pathogen may be alleviated by inclusion of epitopes from multiple antigens from that pathogen in a vaccine composition. A “pathogen” may be an infectious agent or a tumor associated molecule.
  • However, one of the most formidable obstacles to the development of broadly efficacious epitope-based immunotherapeutics has been the extreme polymorphism of HLA molecules. To date, effective non-genetically biased coverage of a population has been a task of considerable complexity; such coverage has required that epitopes be used specific for HLA molecules corresponding to each individual HLA allele, therefore, impractically large numbers of epitopes would have to be used in order to cover ethnically diverse populations. There has existed a need to develop peptide epitopes that are bound by multiple HLA antigen molecules for use in epitope-based vaccines. The greater the number of HLA antigen molecules bound, the greater the breadth of population coverage by the vaccine.
  • Furthermore, as described herein in greater detail, a need has existed to modulate peptide binding properties, for example so that peptides that are able to bind to multiple HLA antigens do so with an affinity that will stimulate an immune response. Identification of epitopes restricted by more than one HLA allele at an affinity that correlates with immunogenicity is important to provide thorough population coverage, and to allow the elicitation of responses of sufficient vigor whereby the natural immune responses noted in self-limiting acute hepatitis, or of spontaneous clearance of chronic HBV infection is induced in a diverse segment of the population. Such a response can also target a broad array of epitopes. The technology disclosed herein provides for such favored immune responses.
  • The information provided in this section is intended to disclose the presently understood state of the art as of the filing date of the present application. Information is included in this section which was generated subsequent to the priority date of this application. Accordingly, background in this section is not intended, in any way, to delineate the priority date for the invention.
  • II. SUMMARY OF THE INVENTION
  • This invention applies our knowledge of the mechanisms by which antigen is recognized by T cells, for example, to develop epitope-based vaccines directed towards HBV. More specifically, this application communicates our discovery of specific epitope pharmaceutical compositions and methods of use in the prevention and treatment of HBV infection.
  • An embodiment of the present invention includes a peptide composition of less than 100 amino acid residues comprising a peptide epitope useful for inducing an immune response against hepatitis B virus (HBV) said epitope (a) having an amino acid sequence of about 8 to about 13 amino acid residues that have at least 65% identity with a native amino acid sequence for HBV, and, (b) binding to at least one MEC class I HLA allele with a dissociation constant of less than about 500 nM. Further, the peptide composition may comprise an amino acid sequence of at least 77% identity, or at least 100% identity with a native HBV amino acid sequence. In a preferred embodiment, the peptide is one of the peptides designated as being from the envelope, polymerase, protein X, or nucleocapsid core regions of HBV. Preferred peptides are described in Tables VI through XVII or XXI.
  • An additional embodiment of the present invention comprises a composition of less than 100 amino acid residues comprising a peptide epitope useful for inducing an immune response against hepatitis B virus (HBV) said peptide (a) having an amino acid sequence of about 8 to about 13 amino acid residues and (b) bearing one of the HLA supermotifs or motifs set out in Tables I and II. Furthermore, the composition may comprise a peptide wherein the peptide is one of those described in Tables VI through XVII or Table XXI which bear an HLA A1, A2, A3, A24, B7, B27, B44, B58, or B62 supermotif; or an HLA A1, A3, A11, A24, or A2.1 motif or an HLA A*3301, A*3101, A*6801, B*0702, B*3501, B51, B*5301, B*5401 motif.
  • In one embodiment of a peptide comprising an HLA A2.1 motif, the peptide does not bear an L or M at position 2 and V at the C-terminal position 9 of a 9 amino acid peptide.
  • An alternative embodiment of the invention comprises an analog of an HBV peptide of less than 100 amino acid residues in length that bears an HLA binding motif, the analog bearing the same HLA binding motif as the peptide but comprising at least one anchor residue that is different from that of the peptide. In a preferred embodiment, said peptide is an analog of a peptide described in Table VI through Table XVII bearing an HLA A1, A2, A3, A24, B7, B27, B44, B58, or B62 supermotif; or an HLA A1, A3, A11, A24, or A2.1 motif or A3301, A3101, A6801, B0702, B3501, B51, B5301, B5401 motif.
  • Embodiments of the invention further include a composition of less than 100 amino acid residues comprising a peptide epitope useful for inducing an immune response against hepatitis B virus (HBV) said peptide (a) having an amino acid sequence of about 9 to about 25 amino acid residues that have at least 65% identity with a native amino acid sequence for HBV and (b) binding to at least one MHC class II HLA allele with a dissociation constant of less than about 1000 nM. In a preferred embodiment, the composition comprises a peptide that has at least 77%, or, 100% identity with a native HBV amino acid sequence. Further, the composition may comprise a peptide wherein said peptide is one of those peptides described in Table XVIII or Table XIX.
  • The invention also includes a peptide composition of less than 100 amino acid residues, said composition comprising an epitope useful for inducing an immune response against hepatitis B virus (HBV) said epitope (a) having an amino acid sequence of about 10 to about 20 amino acid residues and (b) bearing one of the class II HLA motifs set out in Table III. In a preferred embodiment, said peptide is one of those peptides described in Table XVIII or XIX.
  • Additional embodiments of the invention include a composition that comprises an isolated nucleic acid sequence that encodes one of the peptides set out in Tables VI through XIX or XXI or XXIII.
  • Alternatively, an embodiment of the invention comprises a composition that comprises at least two peptides, at least one of said at least two peptides selected from Tables VI-XIX or XXI or XXIII. In a preferred embodiment, two or more of the at least two peptides are depicted in Tables VI-XIX or XXI or XXIII. The composition may further comprise at least one nucleic acid sequence. In a preferred embodiment each of said at least two peptides are encoded by a nucleic acid sequence, wherein each of the nucleic acid sequences are located on a single vector.
  • Embodiments of the invention additionally include a peptide composition of less than 100 amino acid residues, said composition comprising an epitope useful for inducing an immune response against HBV, said epitope having at least one of the amino acid sequences set out in Table XXIII.
  • An alternative modality for defining the peptides in accordance with the invention is to recite the physical properties, such as length; primary, secondary and/or tertiary structure; or charge, which are correlated with binding to a particular allele-specific HLA molecule or group of allele-specific HLA molecules. A further modality for defining peptides is to recite the physical properties of an HLA binding pocket, or properties shared by several allele-specific HLA binding pockets (e.g. pocket configuration and charge distribution) and reciting that the peptide fits and binds to said pocket or pockets.
  • An additional embodiment of the invention comprises a method for inducing a cytotoxic T cell response to HBV in a mammal comprising administering to said mammal at least one peptide from Tables VI to XIX or Table XXI.
  • Further embodiments of the invention include a vaccine for treating HBV infection that induces a protective immune response, wherein said vaccine comprises at least one peptide selected from Tables VI to Table XIX or Table XXI in a pharmaceutically acceptable carrier.
  • Also included as an embodiment of the invention is a vaccine for preventing HBV infection that induces a protective immune response, wherein said vaccine comprises at least one peptide selected from Tables VI to XIX or Table XXI in a pharmaceutically acceptable carrier.
  • The invention further includes an embodiment comprising a method for inducing a cytotoxic T cell response to HBV in a mammal, comprising administering to said mammal a nucleic acid sequence encoding a peptide selected from Tables VI to XIX or Table XXI.
  • A further embodiment of the invention comprises a kit for a vaccine for treating or preventing HBV infection, wherein the vaccine induces a protective immune response, said vaccine comprising at least one peptide selected from Tables VI to XIX or Table XXI in a pharmaceutically acceptable carrier and instructions for administration to a patient.
  • Lastly, the invention includes an embodiment comprising a method for monitoring immunogenic activity of a vaccine for HBV in a patient having a known HLA-type, the method comprising incubating a T lymphocyte sample from the patient with a peptide selected from Tables VI to XIX or Table XXI which binds the product of at least one HLA allele present in said patient, and detecting for the presence of a T lymphocyte that binds to the peptide. In a preferred embodiment, the peptide comprises a tetrameric complex.
  • As will be apparent from the discussion below, other methods and embodiments are also contemplated. Further, novel synthetic peptides produced by any of the methods described herein are also part of the invention.
  • III. BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1: FIG. 1 Illustrates the Position of Peptide Epitopes in Experimental Model Minigene Constructs
  • IV. DETAILED DESCRIPTION OF THE INVENTION
  • The peptides and corresponding nucleic acid compositions of the present invention are useful for stimulating an immune response to HBV either by stimulating the production of CTL or HTL responses. The peptides, which are derived directly or indirectly from native HBV amino acid sequences, are able to bind to HLA molecules and stimulate an immune response to HBV. The complete polyprotein sequence from HBV and its variants can be obtained from Genbank. Peptides can also be readily determined from sequence information that may subsequently be discovered for heretofore unknown variants of HBV as will be clear from the disclosure provided below.
  • The peptides of the invention have been identified in a number of ways, as will be discussed below. Further, analog peptides have been derived and the binding activity for HLA molecules modulated by modifying specific amino acid residues to create peptide analogs exhibiting altered immunogenicity. Further, the present invention provides compositions and combinations of compositions that enable epitope-based vaccines that are capable of interacting with multiple HLA antigens to provide broader population coverage than prior vaccines.
  • The invention can be better understood with reference to the following definitions:
  • IV.A. DEFINITIONS
  • “Cross-reactive binding” indicates that a peptide is bound by more than one HLA molecule; a synonym is degenerate binding.
  • A “cryptic epitope” elicits a response by immunization with an isolated peptide, but the response is not cross-reactive in vitro when intact whole protein which comprises the epitope is used as an antigen.
  • A “dominant epitope” is an epitope that induces an immune response upon immunization with a whole native antigen. (See, e.g., Sercarz, et al., Annu. Rev. Immunol. 11:729766 (1993)) Such a response is cross-reactive in vitro with an isolated peptide epitope.
  • With regard to a particular amino acid sequence, an “epitope” is a set of amino acid residues which is involved in recognition by a particular immunoglobulin, or in the context of T cells, those residues necessary for recognition by T cell receptor proteins and/or Major Histocompatibility Complex (MHC) receptors. In an immune system setting, in vivo or in vitro, an epitope is the collective features of a molecule, such as primary, secondary and tertiary peptide structure, and charge, that together form a site recognized by an immunoglobulin, T cell receptor or HLA molecule.
  • As used herein, “high affinity” with respect to HLA class I molecules is defined as binding with an IC50 (or KD) of less than 50 nM. “Intermediate affinity” is binding with an IC50 (or KD) of between about 50 and about 500 nM. “High affinity” with respect to binding to HLA class II molecules is defined as binding with an KD of less than 100 nM. “Intermediate affinity” is binding with a KD of between about 100 and about 1000 nM. Assays for determining binding are described in detail in PCT publications WO 94/20127 and WO 94/03205. Alternatively, binding is expressed relative to a reference peptide. As a particular assay becomes more, or less, sensitive, the IC50's of the peptides tested may change somewhat. However, the binding relative to the reference peptide will not significantly change. For example, in an assay run under conditions such that the IC50 of the reference peptide increases 10-fold, the IC50 values of the test peptides will also shift approximately 10-fold. Therefore, to avoid ambiguities, the assessment of whether a peptide is a good, intermediate, weak, or negative binder is generally based on its IC50, relative to the IC50 of a standard peptide.
  • “Human Leukocyte Antigen” or “HLA” is a human class I or class II Major Histocompatibility Complex (MEC) protein (see, Stites, et al., IMMUNOLOGY, 8TH ED., Lange Publishing, Los Altos, Calif. (1994).
  • An “HLA supertype or family”, as used herein, describes sets of HLA molecules grouped on the basis of shared peptide-binding specificities. HLA class I molecules that share somewhat similar binding affinity for peptides bearing certain amino acid motifs are grouped into HLA supertypes. The terms HLA superfamily, HLA supertype family, and HLA xx-like supertype molecules (where xx denotes a particular HLA type) are synonyms.
  • Throughout this disclosure, results are expressed in terms of “IC50's.” IC50 is the concentration of peptide in a binding assay at which 50% inhibition of binding of a reference peptide is observed. Given the conditions in which the assays are run (i.e., limiting HLA proteins and labeled peptide concentrations), these values approximate KD values. It should be noted that IC50 values can change, often dramatically, if the assay conditions are varied, and depending on the particular reagents used (e.g., HLA preparation, etc.). For example, excessive concentrations of HLA molecules will increase the apparent measured IC50 of a given ligand.
  • The terms “identical” or percent “identity,” in the context of two or more peptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using a sequence comparison algorithms or by manual alignment and visual inspection.
  • An “immunogenic peptide” or “peptide epitope” is a peptide which comprises an allele-specific motif or supermotif such that the peptide will bind an HLA molecule and induce a CTL and/or HTL response. Thus, immunogenic peptides of the invention are capable of binding to an appropriate HLA molecule and thereafter inducing a cytotoxic T cell response, or a helper T cell response, to the antigen from which the immunogenic peptide is derived.
  • The phrases “isolated” or “biologically pure” refer to material which is substantially or essentially free from components which normally accompany the material as it is found in its native state. Thus, isolated peptides in accordance with the invention preferably do not contain materials normally associated with the peptides in their in situ environment.
  • “Major Histocompatibility Complex” or “MHC” is a cluster of genes that plays a role in control of the cellular interactions responsible for physiologic immune responses. In humans, the MHC complex is also known as the HLA complex. For a detailed description of the MHC and HLA complexes, see, Paul, FUNDAMENTAL IMMUNOLOGY, 3RD ED., Raven Press, New York, 1993.
  • The term “motif” refers to the pattern of residues in a peptide of defined length, usually a peptide of from about 8 to about 13 amino acids for a class I HLA motif and from about 6 to about 25 amino acids for a class II HLA motif, which is recognized by a particular HLA molecule. Peptide motifs are typically different for each protein encoded by each human HLA allele and differ in the pattern of the primary and secondary anchor residues.
  • A “negative binding residue” is an amino acid which if present at certain positions (typically not primary anchor positions) of peptide epitope results in decreased binding affinity of the peptide for the peptide's corresponding HLA molecule.
  • The term “peptide” is used interchangeably with “oligopeptide” in the present specification to designate a series of residues, typically L-amino acids, connected one to the other, typically by peptide bonds between the amino and carboxyl groups of adjacent amino acids. The preferred CTL-inducing oligopeptides of the invention are fewer than 25 residues in length, or less than 15 residues in length or 13 residues or less in length and usually consist of between about 8 and about 11 residues, preferably 9 or 10 residues. The preferred HTL-inducing oligopeptides are less than about 50 residues in length and usually consist of between about 6 and about 30 residues, more usually between about 12 and 25, and often between about 15 and 20 residues.
  • “Pharmaceutically acceptable” refers to a non-toxic, inert, and physiologically compatible composition.
  • A “primary anchor residue” is an amino acid at a specific position along a peptide sequence which is understood to provide a contact point between the immunogenic peptide and the HLA molecule. One to three, usually two, primary anchor residues within a peptide of defined length generally defines a “motif” for an immunogenic peptide. These residues are understood to fit in close contact with peptide binding grooves of an HLA molecule, with their side chains buried in specific pockets of the binding grooves themselves. In one embodiment, the primary anchor residues are located at position 2 (from the amino terminal position) and at the carboxyl terminal position of a 9 residue peptide in accordance with the invention. The primary anchor positions for each motif and supermotif are set forth in Table I. For example, analog peptides can be created by altering the presence or absence of particular residues in these primary anchor positions. Such analogs are used to finely modulate the binding affinity of a peptide comprising a particular motif or supermotif.
  • “Promiscuous binding” is where a distinct peptide is recognized by the same T cell clone in the context of various HLA molecules.
  • A “protective immune response” refers to a CTL and/or an HTL response to an antigen from an infectious agent or a tumor antigen from which an immunogenic peptide is derived, and thereby preventing or at least partially arresting disease symptoms or progression. The immune response may also include an antibody response which has been facilitated by the stimulation of helper T cells.
  • The term “residue” refers to an amino acid or amino acid mimetic incorporated into an oligopeptide by an amide bond or amide bond mimetic.
  • A “secondary anchor residue” is an amino acid at a position other than a primary anchor position in a peptide which may influence peptide binding. A secondary anchor residue occurs at a significantly higher frequency amongst bound peptides than would be expected by random distribution of amino acids at one position. The secondary anchor residues are said to occur at “secondary anchor positions.” A secondary anchor residue can be identified as a residue which is present at a higher frequency among high affinity binding peptides, or a residue otherwise associated with high affinity binding. For example, analog peptides can be created by altering the presence or absence of particular residues in these secondary anchor positions. Such analogs are used to finely modulate the binding affinity of a peptide comprising a particular motif or supermotif.
  • A “subdominant epitope” is an epitope which evokes little or no response upon immunization with whole antigens which comprise the epitope, but for which a response can be obtained by immunization with an isolated peptide, and this response (unlike the case of cryptic epitopes) is detected when whole protein is used to recall the response in vitro or in vivo.
  • A “supermotif” is a peptide binding specificity shared by HLA molecules encoded by two or more HLA alleles. Thus, a preferably is recognized with high or intermediate affinity (as defined herein) by two or more HLA antigens.
  • “Synthetic peptide” refers to a peptide that is not naturally occurring, but is man-made using such methods as chemical synthesis or recombinant DNA technology.
  • The nomenclature used to describe peptide compounds follows the conventional practice wherein the amino group is presented to the left (the N-terminus) and the carboxyl group to the right (the C-terminus) of each amino acid residue. When amino acid residue positions are referred to in a peptide epitope they are numbered in an amino to carboxyl direction with position one being the position closest to the amino terminal. In the formulae representing selected specific embodiments of the present invention, the amino- and carboxyl-terminal groups, although not specifically shown, are in the form they would assume at physiologic pH values, unless otherwise specified. In the amino acid structure formulae, each residue is generally represented by standard three letter or single letter designations. The L-form of an amino acid residue is represented by a capital single letter or a capital first letter of a three-letter symbol, and the D-form for those amino acids having D-forms is represented by a lower case single letter or a lower case three letter symbol. Glycine has no asymmetric carbon atom and is simply referred to as “Gly” or G. Symbols for the amino acids are shown below.
  • Single Three
    Letter Symbol Letter Symbol Amino Acids
    A Ala Alanine
    C Cys Cysteine
    D Asp Aspartic Acid
    E Glu Glutamic Acid
    F Phe Phenylalanine
    G Gly Glycine
    H His Histidine
    I Ile Isoleucine
    K Lys Lysine
    L Leu Leucine
    M Met Methionine
    N Asn Asparagine
    P Pro Proline
    Q Gln Glutamine
    R Arg Arginine
    S Ser Serine
    T Thr Threonine
    V Val Valine
    W Trp Tryptophan
    Y Tyr Tyrosine
  • IV.B. STIMULATION OF CTL AND HTL RESPONSES AGAINST HBV
  • The mechanism by which T cells recognize antigens has been delineated during the past ten years. Based on our new understanding of the immune system we have generated efficacious peptide epitope vaccine compositions that can induce a therapeutic or prophylactic immune response to HBV infection in a broad population. For an understanding of the value and efficacy of the claimed compositions, a brief review of the technology is provided.
  • A complex of an HLA molecule and a peptidic antigen acts as the ligand recognized by HLA-restricted T cells (Buus, S. et al., Cell 47:1071, 1986; Babbitt, B. P. et al., Nature 317:359, 1985; Townsend, A., and Bodmer, H., Annu. Rev. Immunol. 7:601, 1989; Germain, R. N., Annu. Rev. Immunol. 11:403, 1993). Through the study of single amino acid substituted antigen analogs and the sequencing of endogenously bound, naturally processed peptides, critical residues that correspond to motifs required for specific binding to HLA antigen molecules have been identified and are described here and set forth in Tables I, II, and III (see also, e.g., Sette, A. and Grey, H. M., Curr. Opin. Immunol. 4:79, 1992; Sinigaglia, F. and Hammer, J., Curr. Biol. 6:52, 1994; Engelhard, V. H., Curr. Opin. Immunol. 6:13, 1994). Furthermore, x-ray crystallographic analysis of HLA-peptide complexes has revealed pockets within the peptide binding cleft of HLA molecules which accommodate allele-specific residues borne by peptide ligands; these residues in turn determine the HLA binding capacity of the peptides in which they are present (Brown, J. H. et al., Nature 364:33, 1993; Guo, H. C. et al., Proc. Natl. Acad. Sci. USA 90:8053, 1993; Guo, H. C. et al., Nature 360:364, 1992; Silver, M. L. et al., Nature 360:367, 1992; Matsumura, M. et al., Science 257:927, 1992; Madden et al., Cell 70:1035, 1992; Fremont, D. H. et al., Science 257:919, 1992; Saper, M. A., Bjorkman, P. J. and Wiley, D.C., J. Mol. Biol. 219:277, 1991).
  • Accordingly, the definition of class I and class II allele-specific HLA binding motifs or class I supermotifs allows identification of regions within a protein that have the potential of binding particular HLA antigens (see also e.g., Sette, A. and Grey, H. M., Curr. Opin. Immunol. 4:79, 1992; Sinigaglia, F. and Hammer, J., Curr. Biol. 6:52, 1994; Engelhard, V. H., Curr. Opin. Immunol. 6:13, 1994 Kast, W. M. et al., J. Immunol., 152:3904, 1994).
  • Furthermore, a variety of assays to detect and quantify the affinity of interaction between peptide and HLA have also been established (Sette, A. and Grey, H. M., Curr. Opin. Immunol. 4:79, 1992; Sinigaglia, F. and Hammer, J., Curr. Biol. 6:52, 1994; Engelhard, V. H., Curr. Opin. Immunol. 6:13, 1994).
  • We have found that the correlation of binding affinity with immunogenicity is an important factor to be considered when evaluating candidate peptides. Thus, by a combination of motif searches and HLA-peptide binding assays, candidates for epitope-based vaccines have been identified. After determining their binding affinity, additional confirmatory work can be performed to select, amongst these vaccine candidates, epitopes with desired characteristics in terms of antigenicity and immunogenicity. Various strategies can be utilized to evaluate immunogenicity, including:
  • 1) Primary T cell cultures from normal individuals (Wentworth, P. A. et al., Mol. Immunol. 32:603, 1995; Celis, E. et al., Proc. Natl. Acad. Sci. USA 91:2105, 1994; Tsai, V. et al., J. Immunol. 158:1796, 1997; Kawashima, I. et al., Human Immunol. 59:1, 1998); This procedure involves the stimulation of PBL from normal subjects with a test peptide in the presence of antigen presenting cells in vitro over a period of several weeks. T cells specific for the peptide become activated during this time and are detected using a 51 Cr-release assay involving peptide sensitized target cells.
  • 2) Immunization of HLA transgenic mice (Wentworth, P. A. et al., J. Immunol. 26:97, 1996; Wentworth, P. A. et al., Int. Immunol. 8:651, 1996; Alexander, J. et al., J. Immunol. 159:4753, 1997); In this method, peptides in incomplete Freund's adjuvant are administered subcutaneously to HLA transgenic mice. Several weeks following immunization, splenocytes are removed and cultured in vitro in the presence of test peptide for approximately one week. Peptide-specific T cells are detected using a 51Cr-release assay involving peptide sensitized target cells and target cells expressing endogenously generated antigen.
  • 3) Demonstration of recall T cell responses from immune individuals who have recovered from infection, and/or from chronically infected patients (Rehermann, B. et al., J. Exp. Med. 181:1047, 1995; Doolan, D. L. et al., Immunity 7:97, 1997; Bertoni, R. et al., J. Clin. Invest. 100:503, 1997; Threlkeld, S. C. et al., J. Immunol. 159:1648, 1997; Diepolder, H. M. et al., J. Virol. 71:6011, 1997). In applying this strategy, recall responses were detected by culturing PBL from subjects that had been naturally exposed to the antigen, for instance through infection, and thus had generated an immune response “naturally”. PBL from subjects were cultured in vitro for 1-2 weeks in the presence of test peptide plus antigen presenting cells (APC) to allow activation of “memory” T cells, as compared to “naive” T-cells. At the end of the culture period, T cell activity is detected using assays for T cell activity including 51Cr release involving peptide-sensitized targets, T cell proliferation or lymphokine release.
  • The following describes the peptide epitopes and corresponding nucleic acids of the invention.
  • IV.C. IMMUNE RESPONSE STIMULATING PEPTIDES
  • As indicated herein, the large degree of HLA polymorphism is an important factor to be taken into account with the epitope-based approach to vaccine development. To address this factor, epitope selection encompassing identification of peptides capable of binding at high or intermediate affinity to multiple HLA molecules is preferably utilized, most preferably these epitopes bind at high or intermediate affinity to two or more allele specific HLA molecules.
  • IV.C.1. Binding Affinity of the Peptides for HLA Molecules
  • CTL-inducing peptides of interest for vaccine compositions preferably include those that have a binding affinity for class I HLA molecules of less than 500 nM. HTL-inducing peptides preferably include those that have a binding affinity for class II HLA molecules of less than 1000 nM. For example, peptide binding is assessed by testing the capacity of a candidate peptide to bind to a purified HLA molecule in vitro. Peptides exhibiting high or intermediate affinity are then considered for further analysis. Selected peptides are tested on other members of the supertype family. In preferred embodiments, peptides that exhibit cross-reactive binding preferably are then used in cellular screening analyses. A peptide is considered to be an epitope if it possesses the molecular features that form the binding site for a particular immunoglobulin or T cell receptor protein.
  • As disclosed herein, high HLA binding affinity is correlated with greater immunogenicity. Greater immunogenicity can be manifested in several different ways. Immunogenicity corresponds to whether an immune response is elicited at all, and to the vigor of any particular response. For example, a peptide might elicit an immune response in a diverse array of the population, yet in no instance produce a vigorous response. In accordance with these principles, close to 90% of high binding peptides have been found to be immunogenic, as contrasted with about 50% of the peptides which bind with intermediate affinity. Moreover, higher binding affinity peptides leads to more vigorous immunogenic responses. As a result, less peptide is required to elicit a similar biological effect if a high affinity binding peptide is used. Thus, in preferred embodiments of the invention, high binding epitopes are particularly desired.
  • The relationship between binding affinity for HLA class I molecules and immunogenicity of discrete peptide epitopes on bound antigens has been determined for the first time in the art by the present inventors. The correlation between binding affinity and immunogenicity was analyzed in two different experimental approaches (Sette, et al., J. Immunol. 153:5586-5592, 1994). In the first approach, the immunogenicity of potential epitopes ranging in HLA binding affinity over a 10,000-fold range was analyzed in HLA-A*0201 transgenic mice. In the second approach, the antigenicity of approximately 100 different hepatitis B virus (HBV)-derived potential epitopes, all carrying A*0201 binding motifs, was assessed by using PBL (peripheral blood lymphocytes) of acute hepatitis patients. Pursuant to these approaches, it was determined that an affinity threshold of approximately 500 nM (preferably 500 nM or less) determines the capacity of a peptide epitope to elicit a CTL response. These data are true for class I binding affinity measurements for naturally processed peptides and for synthesized T cell epitopes. These data also indicate the important role of determinant selection in the shaping of T cell responses.
  • An affinity threshold associated with immunogenicity in the context of HLA class II DR molecules has also been delineated (Southwood et al. J. Immunology 160:3363-3373, 1998, and U.S. Ser. No. 60/087,192 filed May 29, 1998). In order to define a biologically significant threshold of DR binding affinity, a database of the binding affinities of 32 DR-restricted epitopes for their restricting element was compiled. In approximately half of the cases (15 of 32 epitopes), DR restriction was associated with high binding affinities, i.e. binding affinities of less than 100 nM. In the other half of the cases (16 of 32), DR restriction was associated with intermediate affinity (binding affinities in the 100-1000 nM range). In only one of 32 cases was DR restriction associated with an IC50 of 1000 nM or greater. Thus, 1000 nM can be defined as an affinity threshold associated with immunogenicity in the context of DR molecules.
  • The binding affinity of peptides for HLA molecules can be determined as described in Example 1, below.
  • IV.C.2. Peptide Binding Motifs and Supermotifs
  • In the past few years evidence has accumulated to demonstrate that a large fraction of HLA class I, and possibly class II molecules can be classified into a relatively few supertypes characterized by largely overlapping peptide binding repertoires, and consensus structures of the main peptide binding pockets. Through the study of single amino acid substituted antigen analogs and the sequencing of endogenously bound, naturally processed peptides, critical residues required for allele-specific binding to HLA molecules have been identified. These motifs are relevant since they indicate peptides that have binding affinity for HLA molecules.
  • For HLA molecule pocket analyses, the residues comprising the B and F pockets of HLA class I molecules as described in crystallographic studies (Guo, H. C. et al., Nature 360:364, 1992; Saper, M. A., Bjorkman, P. J. and Wiley, D.C., J. Mol. Biol. 219:277, 1991; Madden, D. R., Garboczi, D. N. and Wiley, D.C., Cell 75:693, 1993), have been compiled from the database of Parham, et al. (Parham, P., Adams, E. J., and Arnett, K. L., Immunol. Rev. 143:141, 1995). In these analyses, residues 9, 45, 63, 66, 67, 70, and 99 were considered to make up the B pocket, and to determine the specificity for the residue in the second position of peptide ligands. Similarly, residues 77, 80, 81, and 116 were considered to determine the specificity of the F pocket, and to determine the specificity for the C-terminal residue of a peptide ligand bound by the HLA molecule.
  • Peptides of the present invention may also include epitopes that bind to MHC class II DR molecules. A significant difference between class I and class II HLA molecules is that, although a stringent size restriction exists for peptide binding to class I molecules, a greater degree of heterogeneity in both sizes and binding frame positions of the motif, relative to the N and C termini of the peptide, can be demonstrated for class II peptide ligands. This increased heterogeneity is due to the structure of the class II-binding groove which, unlike its class I counterpart, is open at both ends. Crystallographic analysis of DRB*0101-peptide complexes (see, e.g., Madden, D. R. Ann. Rev. Immunol. 13:587 (1995)) showed that the residues occupying position 1 and position 6 of peptides complexed with DRB*0101 engage two complementary pockets on the DRBa*0101 molecules, with the P1 position corresponding to the most crucial anchor residue and the deepest hydrophobic pocket. Other studies have also pointed to the P6 position as a crucial anchor residue for binding to various other DR molecules.
  • Thus, peptides of the present invention are identified by any one of several HLA-specific amino acid motifs. If the presence of the motif corresponds to the ability to bind several allele-specific HLA antigens it is referred to as a supermotif. The allele-specific HLA molecules that bind to peptides that possess a particular amino acid supermotif are collectively referred to as an HLA “supertype.”
  • The peptide motifs and supermotifs described below provide guidance for the identification and use of peptides in accordance with the invention. Examples of peptide epitopes bearing the respective supermotif or motif are included in Tables as designated in the description of each motif or supermotif. The Tables include a binding affinity ratio listing for some of the peptide epitopes. The ratio may be converted to IC50 by using the following formula: IC50 of the standard peptide/ratio=IC50 of the test peptide (i.e. the peptide epitope). The IC50 values of standard peptides used to determine binding affinities for Class I peptides are shown in Table IV. The IC50 values of standard peptides used to determine binding affinities for Class II peptides are shown in Table V. The peptides used as standards for the binding assay are examples of standards; alternative standard peptides can also be used when performing such an analysis.
  • To obtain the peptide epitope sequences listed in each Table, protein sequence data from twenty HBV strains (HPBADR, HPBADR1CG, HPBADRA, HPBADRC, HPBADRCG, HPBCGADR, HPBVADRM, HPBADW, HPBADW1, HPBADW2, HPBADW3, HPBADWZ, HPBHEPB, HPBVADW2, HPBADR, HPBV, HPBVAYWC, HPBVAYWCI, NAD HPBVAYWE) were evaluated for the presence of the designated supermotif or motif. Peptide epitopes were also selected on the basis of their conservancy. A criterion for conservancy requires that the entire sequence of a peptide be totally conserved in 75% of the sequences available for a specific protein. The percent conservancy of the selected peptide epitopes is indicated on the Tables. The frequency, i.e. the number of strains of the 20 strains in which the peptide sequence was identified, is also shown. The “1st position” column in the Tables designates the amino acid position of the HBV polyprotein that corresponds to the first amino acid residue of the epitope. Preferred peptides are designated by an asterisk.
  • HLA Class I Motifs Indicative of CTL Inducing Peptide Epitopes: IV.C.2.a) HLA-A1 Supermotif
  • The HLA-A1 supermotif is characterized by peptides having a general motif of small (T or S) and hydrophobic (L, I, V, M, or F) primary anchor residues in position 2, and aromatic (Y, F, or W) primary anchor residues at the C-terminal position The corresponding family of HLA molecules that bind to the A1 supermotif (the HLA-A1 supertype) includes A*0101, A*2601, A*2602, A*2501, and A*3201. (DiBrino, M. et al., J. Immunol. 151:5930, 1993; DiBrino, M. et al., J. Immunol. 152:620, 1994; Kondo, A. et al., Immunogenetics 45:249, 1997; Dumrese et al., submitted). Peptides binding to each of the individual HLA proteins can be modulated by substitutions at primary anchor positions.
  • Representative peptide epitopes that contain the A1 supermotif are set forth on the attached Table VI.
  • IV.C.2.b) HLA-A2 Supermotif
  • The HLA-A2 supermotif is characterized by the presence in peptide ligands of small or aliphatic amino acids (L, I, V, M, A, T, or Q) at position 2 and L, I, V, M, A, or T at the C-terminal position. These positions are referred to as primary anchors. The corresponding family of HLA molecules (the HLA-A2 supertype that binds these peptides) is comprised of at least nine HLA-A proteins: A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*0207, A*6802, and A*6901. As explained in detail below, binding to each of the individual allele-specific HLA molecules can be modulated by substitutions at the primary anchor and/or secondary anchor positions.
  • Representative peptide epitopes that contain the A2 supermotif are set forth on the attached Table VII.
  • IV.C.2.c) HLA-A3 Supermotif
  • The HLA-A3 supermotif is characterized by peptide ligands having primary anchor residues: A, L, I, V, M, S, or, T at position 2, and positively charged residues, such as R or K at the C-terminal position (in position 9 of 9-mers). Exemplary members of the corresponding HLA family of HLA molecules (the HLA-A3 superfamily) that bind the A3 supermotif include: A3 (A*0301), A11 (A*1101), A31 (A*3101), A*3301, and A*6801. Other allele-encoded HLA molecules predicted to be members of the A3 superfamily include A34, A66, and A*7401. As explained in detail below, peptide binding to each of the individual allele-specific HLA proteins can be modulated by substitutions of amino acids at the primary and/or secondary anchor positions of the peptide.
  • Representative peptide epitopes that contain the A3 supermotif are set forth on the attached Table VIII.
  • IV.C.2.d) HLA-A24 Supermotif
  • The HLA-A24 supermotif is characterized by the presence in peptide ligands of an aromatic (F, W, or Y) residue as a primary anchor in position 2 and a hydrophobic (Y, F, L, I, V, or M) residue as primary anchor at the C-terminal position. The corresponding family of HLA molecules that bind to the A24 supermotif (the A24 supertype) includes A*2402, A*3001, and A*2301. Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary anchor positions.
  • Representative peptide epitopes that contain the A24 supermotif are set forth on the attached Table IX.
  • IV.C.2.e) HLA-B7 Supermotif
  • The HLA-B7 supermotif is characterized by peptides bearing proline in position 2 as a primary anchor and hydrophobic or aliphatic amino acids (L, I, V, M, A, F, W, or Y) as the primary anchor at the C-terminal position. The corresponding family of HLA molecules that bind the B7 supermotif (the HLA-B7 supertype) is comprised of at least a dozen HLA-B proteins including B7, B*3501-1, B*3502-2, B*3501-3, B51, B*5301, B*5401, B*5501, B*5401, B*5501, B*5502, B*5601, B*6701, and B*7801 (See, e.g., Sidney, et al., J. Immunol. 154:247 (1995); Barber, et al., Curr. Biol. 5:179 (1995); Hill, et al., Nature 360:434 (1992); Rammensee, et al., Immunogenetics 41:178 (1995)). As explained in detail below, peptide binding to each of the individual allele-specific HLA proteins can be modulated by substitutions at the primary and/or secondary anchor positions of the peptide.
  • Representative peptide epitopes that contain the B7 supermotif are set forth on the attached Table X.
  • IV.C.2.f) HLA-B27 Supermotif
  • The HLA-B27 supermotif is characterized by the presence in peptide ligands of positively charged (R, H, or K) residues as primary anchors at position 2 and hydrophobic (A, L, I, V, M, Y, F, or W) residues as primary anchors at the C-terminal. Exemplary members of the corresponding HLA molecules that bind to the B27 supermotif (the B27 supertype) include B*14, B*1509, B*38, B*3901, B*3902, B*73, and various B27 subtypes. Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary anchor positions.
  • Representative peptide epitopes that contain the B27 supermotif are set forth on the attached Table XI.
  • IV.C.2.g) HLA-B44 Supermotif
  • The HLA-B44 supermotif is characterized by the presence in peptide ligands of negatively charged (D or E) residues as a primary anchor in position 2, and hydrophobic residues (F, W, Y, L, I, M V, or A) as a primary anchor at the C-terminal. Exemplary members of the corresponding family of HLA molecules that bind to the B44 supermotif (the B44 supertype) include B*3701, B*4402, B*4403, B60, and B61. Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary anchor positions.
  • Representative peptide epitopes that contain the B44 supermotif are set forth on the attached Table XII.
  • IV.C.2.h) HLA-B58 Supermotif
  • The HLA-B58 supermotif is characterized by the presence in peptide ligands of small aliphatic residues (A, S, or T) as primary anchor residues at position 2 and aromatic or hydrophobic residues (F, W, Y, L, I, or V) as primary anchor residues at the C-terminal. Exemplary members of the corresponding HLA molecules that bind to the B58 supermotif (the B58 supertype) include B*1516, B*1517, B*5701, B*5702, and B*58. Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary anchor positions.
  • Representative peptide epitopes that contain the B58 supermotif are set forth on the attached Table XIII.
  • IV.C.2.i) HLA-B62 Supermotif
  • The HLA-B62 supermotif is characterized by the presence in peptide ligands of the polar aliphatic residue Q or the hydrophobic aliphatic residues (L, V, M, or I) as a primary anchor in position 2 and hydrophobic residues (F, W, Y, M, I, or V) as a primary anchor at the C-terminal position. Exemplary members of the corresponding HLA molecules that a bind to the B62 supermotif (the B62 supertype) include B46, B52, B62, B75, and B77. Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary anchor positions.
  • Representative peptide epitopes that contain the B62 supermotif are set forth on the attached Table XIV.
  • IV.C.2.j) HLA-A1 Motif
  • The allele-specific HLA-A1 motif is characterized by the presence in peptide ligands of T, S, or M as a primary anchor residue at position 2 and the presence of Y as a primary anchor residue at the C-terminal position. Alternatively, a primary anchor residue may be present at position 3 rather than position 2. This motif is characterized by the presence of D, E, A, or S as a primary anchor residue in position 3 and a Y as a primary anchor residue at the C-terminus. Peptide binding to HLA A1 can be modulated by substitutions at primary and/or secondary anchor positions.
  • Representative peptide epitopes that contain the A1 motif are set forth on the attached Table XV.
  • IV.C.2.k) HLA-A3 Motif
  • The allele-specific HLA-A3 motif is characterized by the presence in peptide ligands of L, M, V, I, S, A, T, F, C, G, or D as a primary anchor residue at position 2 and the presence of K, Y, R, H, F, or A as the primary anchor residue at the C-terminal position. Peptide binding to HLA-A3 can be modulated by substitutions at primary and/or secondary anchor positions.
  • Representative peptide epitopes that contain the A3 motif are set forth on the attached Table XVI.
  • IV.C.2.l) HLA-A11 Motif
  • The allele-specific HLA-A11 motif is characterized by the presence in peptide ligands of V, T, M, L, I, S, A, G, N, C, D, or F as a primary anchor residue in position 2 and K, R, Y, or H as a primary anchor residue at the C-terminal position. Peptide binding to HLA-A11 can be modulated by substitutions at primary and/or secondary anchor positions.
  • Representative peptide epitopes that contain the A11 motif are set forth on the attached Table XVI; peptides bearing the A3 allele-specific motif are also present in Table XVII. The A11 and A3 motifs have a number of anchor residues in common, separate tables would provide a number of redundant entries.
  • IV.C.2.m) HLA-A24 Motif
  • The allele-specific HLA-A24 motif is characterized by the presence in peptide ligands of Y, F, W, or M as a primary anchor residue in position 2 and F, L, I, or W as a primary anchor residue at the C-terminal position. Peptide binding to HLA-A24 molecules can be modulated by substitutions at primary and/or secondary anchor positions.
  • Representative peptide epitopes that contain the A24 motif are set forth on the attached Table XVII.
  • IV.C.2.n) HLA-A2.1 Motif
  • The allele-specific HLA-A2.1 motif was first determined to be characterized by the presence in peptide ligands of L, M, V, I, A or T as a primary anchor residue in position 2 and, L, V, I, A, or T as a primary anchor residue at the C-terminal position. The preferred and tolerated residues that characterize the primary anchor positions of the HLA-A2.1 motif are identical to the preferred residue of the A2 supermotif. Secondary anchor residues that characterize the A2.1 motif have additionally been defined as disclosed herein. These are disclosed in Table II. Peptide binding to HLA-A2.1 molecules can be modulated by substitutions at primary and/or secondary anchor positions.
  • Representative peptide epitopes that contain the A2.1 motif are set forth on the attached Table VII. These peptides, which bear the HLA-A2 supermotif, also contain secondary anchor residues that are characteristic of the HLA-A2.1 motif. In one embodiment, the peptide epitope does not bear an L or M at position 2 and V at the C-terminal position 9 of a 9-amino acid peptide.
  • The primary anchor residues of the HLA class I peptide epitope supermotifs and motifs delineated above are summarized in Table I. Primary and secondary anchor positions are summarized in Table II.
  • Motifs Indicative of Class II HTL Inducing Peptide Epitopes IV.C.2.o) HLA DR-1-4-7 Supermotif
  • Motifs have also been identified for peptides that bind to three common HLA class II types, HLA DRB1*0401, DRB1*0101, and DRB1*0701. Peptides binding to these DR molecules carry a motif characterized by a large aromatic or hydrophobic residue in position 1 (Y, F, W, L, I, V, or M) and a small, non-charged residue in position 6 (S, T, C, AP, V, I, L, or M). Allele specific secondary effects and secondary anchors for each of these HLA types have also been identified. These are set forth in Table III. Peptide binding to HLA-DR4, DR1, and DR7 can be modulated by substitutions at primary and/or secondary anchor positions.
  • Representative peptides are set forth in Table XVIII.
  • IV.C.2.p) HLA DR3 Motifs
  • Two alternative motifs characterize peptides that bind to HLA-DR3 molecules. In the first motif, a large, hydrophobic residue (I, L, V, M, Y, or F) is present in anchor position 1 and D is present as an anchor at position 4, which is defined as being 3 positions from anchor position 1 towards the carboxyl terminus regardless of the location of anchor position 1 in the peptide. Lack of either the large, hydrophobic residue at anchor position 1, or of the negatively charged or amide-like anchor residue at position 4 may be compensated for by the presence of a positive charge at position 6 (which is defined as being 5 positions from anchor position 1 towards the carboxyl terminus). Thus for the second, alternative motif I, L, V, M, Y, F, or A is present at anchor position 1; D, N, Q, E, S, or T is present at anchor position 4; and K, R, or H is present at anchor position 6. Peptide binding to HLA-DR3 can be modulated by substitutions at primary and/or secondary anchor positions.
  • Representative peptides are set forth in Table IXX.
  • IV.C.3. Enhancing Population Coverage of the Vaccine
  • Vaccines that have broad population coverage are preferred because they are more commercially viable and generally applicable to the most people. Broad population coverage can be obtained using the peptides of the invention (and nucleic acid compositions that encode such peptides) through selecting peptide epitopes that bind to HLA alleles which, when considered in total, are present in most of the population. Table XX lists the overall frequencies of the A2-, A3-, and B7-supertypes in various ethnicities. Coverage in excess of 80% is achieved with these motifs. These results suggest that effective and non-ethnically biased population coverage is achieved upon use of a limited number of cross-reactive peptides. Although the population coverage reached with these three main peptide specificities is high, coverage can be expanded to reach 95% population coverage and above, and more easily achieve truly multispecific responses upon use of additional supermotif or allele-specific motif bearing peptides.
  • Table XX summarizes the HLA supertypes that have been identified, and indicates an estimate of their combined prevalence in major ethnic groups. The B44-, A1-, and A24-supertypes are present, on average, in over 25% of the world's major ethnic populations. While less prevalent overall, the B27-, B58-, and B62 supertypes are each present with a frequency >25% in at least one major ethnic group. The Table indicates the population coverage achieved by the A2-, A3-, and B7-supertypes, and the incremental coverage obtained by the addition of A1-, A24-, and B44-supertypes, or all of the supertypes described herein. As shown, by including epitopes from the six most frequent supertypes, an average population coverage of 99% is obtained for five major ethnic groups.
  • The data presented herein, together with the previous definition of the A2-, A3-, and B7-supertypes, indicates that all antigens, with the possible exception of A29, B8, and B46, can be classified into a total of nine HLA supertypes. Focusing on the six most common supertypes affords population coverage greater than 98% for all major ethnic populations.
  • IV.D. IMMUNE RESPONSE STIMULATING PEPTIDE ANALOGS
  • Although peptides with suitable cross-reactivity among all alleles of a superfamily are identified by the screening procedures described above, cross-reactivity is not always complete and in such cases procedures to further increase cross-reactivity of peptides can be useful; such procedures can also be used to modify other properties of the peptides. Having established the general rules that govern cross-reactivity of peptides for HLA alleles within a given motif or supermotif, modification (i.e., analoging) of the structure of peptides of particular interest in order to achieve broader (or otherwise modified) HLA binding capacity can be performed. More specifically, peptides which exhibit the broadest cross-reactivity patterns, (both amongst the known T cell epitopes, as well as the more extended set of peptides that contain the appropriate supermotifs), can be produced in accordance with the teachings herein.
  • The strategy employed utilizes the motifs or supermotifs which correlate with binding to certain HLA molecules. The motifs or supermotifs are defined by having primary anchors, though secondary anchors can also be modified. Analog peptides can be created by substituting amino acids residues at primary anchor, secondary anchor, or at primary and secondary anchor positions. Generally, analogs are made for peptides that already bear a motif or supermotif. Preferred secondary anchor residues of supermotifs and motifs that have been defined for HLA class I and class II binding peptides are shown in Tables II and III, respectively.
  • For a number of the motifs or supermotifs in accordance with the invention, residues are defined which are deleterious to binding to allele-specific HLA molecules or members of HLA supertypes that bind to the respective motif or supermotif (Tables II and III). Accordingly, removal of residues that are detrimental to binding can be performed in accordance with the present invention. For example, in the case of the A3 supertype, when all peptides that have such deleterious residues are removed from the population of analyzed peptides, the incidence of cross-reactivity increases from 22% to 37% (see, e.g., Sidney, J. et al., Hu. Immunol. 45:79, 1996). Thus, one strategy to improve the cross-reactivity of peptides within a given supermotif is simply to delete one or more of the deleterious residues present within a peptide and substitute a small “neutral” residue such as Ala (that may not influence T cell recognition of the peptide). An enhanced likelihood of cross-reactivity is expected if, together with elimination of detrimental residues within a peptide, residues associated with high affinity binding to multiple alleles within a superfamily are inserted.
  • To ensure that changes in the native or original epitope recognized by T cells do not lead to a failure of killing antigen presenting cells presenting the unaltered “wild type” peptide (or, in the case of class II epitopes, a failure to elicit helper T cells that cross-react with the wild type peptides), the variant peptide may be used to immunize T cells in vitro from individuals of the appropriate HLA allele, and the cells' capacity to induce lysis of wild type peptide sensitized target cells is evaluated. In both class I and class II systems it will be desirable to use as targets, cells that have been either infected or transfected with the appropriate genes to establish whether endogenously produced antigen is also recognized by the relevant T cells.
  • Another embodiment of the invention to ensure adequate numbers of cross-reactive cellular binders is to create analogs of weak binding peptides. Class I peptides exhibiting binding affinities of 500-50000 nM, and carrying an acceptable but suboptimal primary anchor residue at one or both positions can be “fixed” by substituting preferred anchor residues in accordance with the respective supertype. The analog peptides can then be tested for crossbinding activity.
  • Another embodiment for generating effective peptide analogs involves the substitution of residues that have an adverse impact on peptide stability or solubility in a liquid environment. This substitution may occur at any position of the peptide epitope. For example, a cysteine (C) can be substituted out in favor of α-amino butyric acid. Due to its chemical nature, cysteine has the propensity to form disulfide bridges and sufficiently alter the peptide structurally so as to reduce binding capacity. Substituting α-amino butyric acid for C not only alleviates this problem, but actually improves binding and crossbinding capability in certain instances (Review: A. Sette et al, In: Persistent Viral Infections, Eds. R. Ahmed and I. Chen, John Wiley & Sons, England, in press, 1998). Substitution of cysteine with α-amino butyric acid may occur at any residue of a peptide epitope, i.e. at either anchor or non-anchor positions.
  • In general, CTL and HTL responses are not directed against all possible epitopes. Rather, they are restricted to a few immunodominant determinants (Zinkernagel, et al., Adv. Immunol. 27:5159, 1979; Bennink, et al., J. Exp. Med. 168:19351939, 1988; Rawle, et al., J. Immunol. 146:3977-3984, 1991). It has been recognized that immunodominance (Benacerraf, et al., Science 175:273-279, 1972) could be explained by either the ability of a given epitope to selectively bind a particular HLA protein (determinant selection theory) (Vitiello, et al., J. Immunol. 131:1635, 1983); Rosenthal, et al., Nature 267:156-158, 1977), or being selectively recognized by the existing TCR (T cell receptor) specificity (repertoire theory) (Klein, J., IMMUNOLOGY, THE SCIENCE OF SELFNONSELF DISCRIMINATION, John Wiley & Sons, New York, pp. 270-310, 1982). It has been demonstrated that additional factors, mostly linked to processing events, can also play a key role in dictating, beyond strict immunogenicity, which of the many potential determinants will be presented as immunodominant (Sercarz, et al., Annu. Rev. Immunol. 11:729-766, 1993).
  • The concept of dominance and subdominance is relevant to immunotherapy of both infectious diseases and cancer. For example, in the course of chronic viral disease, recruitment of subdominant epitopes can be important for successful clearance of the infection, especially if dominant CTL or HTL specificities have been inactivated by functional tolerance, suppression, mutation of viruses and other mechanisms (Franco, et al., Curr. Opin. Immunol. 7:524-531, (1995)). In the case of cancer and tumor antigens, CTLs recognizing at least some of the highest binding affinity peptides might be functionally inactivated. Lower binding affinity peptides are preferentially recognized at these times.
  • In particular, it has been noted that a significant number of epitopes derived from known non-viral tumor associated antigens (TAA) bind HLA class I with intermediate affinity (IC50 in the 50-500 nM range). For example, it has been found that 8 of 15 known TAA peptides recognized by tumor infiltrating lymphocytes (TIL) or CTL bound in the 50-500 nM range. (These data are in contrast with estimates that 90% of known viral antigens that were recognized as peptides bound HLA with IC50 of 50 nM or less, while only approximately 10% bound in the 50-500 nM range (Sette, et al., J. Immunol., 153:558-5592 (1994)). In the cancer setting this phenomenon is probably due to elimination, or functional inhibition of the CTL recognizing several of the highest binding peptides, presumably because of T cell tolerization events.
  • Without intending to be bound by theory, it is believed that because T cells to dominant epitopes may have been clonally deleted, selecting subdominant epitopes may allow extant T cells to be recruited, which will then lead to a therapeutic response. However, the binding of HLA molecules to subdominant epitopes is often less vigorous than to dominant ones. Accordingly, there is a need to be able to modulate the binding affinity of particular immunogenic epitopes for one or more HLA molecules, and thereby to modulate the immune response elicited by the peptide. Thus a need exists to prepare analog peptides which elicit a more vigorous response. This ability would greatly enhance the usefulness of peptide-based vaccines and therapeutic agents.
  • Representative analog peptides are set forth in Table XXI. The Table indicates the length and sequence of the analog peptide as well as the motif or supermotif, if appropriate. The term “fixed nomenclature” refers to the change
  • IV.E. COMPUTER SCREENING OF PROTEIN SEQUENCES FROM DISEASE-RELATED ANTIGENS FOR SUPERMOTIF OR MOTIF CONTAINING PEPTIDES
  • Computer programs that allow the rapid screening of protein sequences for the occurrence of the subject supermotifs or motifs are encompassed by the present invention; as are programs that permit the generation of analog peptides. These programs are implemented to analyze any identified amino acid sequence or operate on an unknown sequence and simultaneously determine the sequence and identify motif-bearing epitopes thereof; analogs can be simultaneously determined as well. Generally, the identified sequences will be from a pathogenic organism or a tumor-associated peptide. For example, the target molecules considered herein include all of the HBV proteins (e.g. surface, core, polymerase, and X).
  • In cases where the sequence of multiple variants of the same target protein are available, peptides are also selected on the basis of their conservancy. A presently preferred criterion for conservancy defines that the entire sequence of a peptide be totally conserved in 75% of the sequences evaluated for a specific protein; this definition of conservancy has been employed herein.
  • It is important that the selection criteria utilized for prediction of peptide binding are as accurate as possible, to correlate most efficiently with actual binding. Prediction of peptides that bind, for example, to HLA-A*0201, on the basis of the presence of the appropriate primary anchors, is positive at about a 30% rate (Ruppert, J. et al. Cell 74:929, 1993). However, by analyzing an extensive peptide-HLA binding database, the present inventors have developed a number of allele specific polynomial algorithms that dramatically increase the predictive value over identification on the basis of the presence of primary anchor residues alone. These algorithms take into account not only the presence or absence of the correct primary anchors, but also consider the positive or deleterious presence of secondary anchor residues (to account for the impact of different amino acids at different positions). The algorithms are essentially based on the premise that the overall affinity (or AG) of peptide-HLA interactions can be approximated as a linear polynomial function of the type:

  • ΔG=a 1i ×a 2i ×a 3i . . . ×a ni
  • where aij is a coefficient that represents the effect of the presence of a given amino acid (j) at a given position (i) along the sequence of a peptide of n amino acids. An important assumption of this method is that the effects at each position are essentially independent of each other. This assumption is justified by studies that demonstrated that peptides are bound to HLA molecules and recognized by T cells in essentially an extended conformation. Derivation of specific algorithm coefficients has been described in Gulukota et al. (Gulukota, K. et al., J. Mol. Biol. 267:1258, 1997).
  • Additional methods to identify preferred peptide sequences, which also make use of specific motifs, include the use of neural networks and molecular modeling programs (Gulukota, K. et al., J. Mol. Biol. 267:1258, 1997; Milik et al., Nature Biotechnology 16:753, 1998; Altuvia et al., Hum. Immunol. 58:1, 1997; Altuvia et al, J. Mol. Biol. 249:244, 1995).
  • For example, it has been shown that in sets of A*0201 motif peptides, 69% of the peptides containing at least one preferred secondary anchor residue while avoiding the presence of any deleterious secondary anchor residues, will bind A*0201 with an IC50 less than 500 nM (Ruppert, J. et al. Cell 74:929, 1993). These algorithms are also flexible in that cut-off scores may be adjusted to select sets of peptides with greater or lower predicted binding properties, as desired.
  • In utilizing computer screening to identify peptide epitopes, all protein sequence or translated sequence may be analyzed using software developed to search for motifs, for example the “FINDPATTERNS” program (Devereux, et al. Nucl. Acids Res. 12:387-395, 1984) or MotifSearch 1.4 software program (D. Brown, San Diego, Calif.) to identify potential peptide sequences containing appropriate HLA binding motifs. As appreciated by one of ordinary skill in the art a large array of software and hardware options are available which can be employed to implement the motifs of the invention relative to known or unknown peptide sequences. The identified peptides will then be scored using customized polynomial algorithms to predict their capacity to bind specific HLA class I or class II alleles.
  • In accordance with the procedures described above, HBV peptides and analogs thereof that are able to bind HLA supertype groups or allele-specific HLA molecules have been identified (Tables VI-XIX; Table XXI).
  • IV.F. ASSAYS TO DETECT T-CELL RESPONSES
  • Once HLA binding peptides are identified, they can be tested for the ability to elicit a T-cell response. The preparation and evaluation of motif-bearing peptides are described in PCT publications WO 94/20127 and WO 94/03205. Briefly, peptides comprising epitopes from a particular antigen are synthesized and tested for their ability to bind to the appropriate HLA proteins in assays using, for example, purified HLA class I molecules and radioiodonated peptides and/or cells expressing empty class I molecules (which lack peptide in their receptor) by, for instance, immunofluorescent staining and flow microfluorimetry, peptide-dependent class I assembly assays, and inhibition of CTL recognition by peptide competition. Those peptides that bind to the class I molecule are further evaluated for their ability to serve as targets for CTLs derived from infected or immunized individuals, as well as for their capacity to induce primary in vitro or in vivo CTL responses that can give rise to CTL populations capable of reacting with selected target cells associated with a disease. Corresponding assays are used for evaluation of HLA class II binding peptides.
  • Conventional assays utilized to detect CTL responses include proliferation assays, lymphokine secretion assays, direct cytotoxicity assays, and limiting dilution assays. For example, antigen-presenting cells that have been incubated with a peptide can be assayed for the ability to induce CTL responses in responder cell populations. Antigen-presenting cells can be normal cells such as peripheral blood mononuclear cells or dendritic cells. Alternatively, mutant mammalian cell lines that are deficient in their ability to load class I molecules with internally processed peptides and that have been transfected with the appropriate human class I gene may be used to test for the capacity of the peptide to induce in vitro primary CTL responses.
  • Peripheral blood lymphocytes may be used as the responder cell source of CTL precursors. The appropriate antigen-presenting cells are incubated with peptide and the peptide-loaded antigen-presenting cells are then incubated with the responder cell population under optimized culture conditions. Positive CTL activation can be determined by assaying the culture for the presence of CTLs that kill radio-labeled target cells, both specific peptide-pulsed targets as well as target cells expressing endogenously processed forms of the HBV antigen from which the peptide sequence was derived.
  • More recently, a method has also been devised which allows direct quantification of antigen-specific T cells by staining with Fluorescein-labeled HLA tetrameric complexes (Altman, J. D. et al., Proc. Natl. Acad. Sci. USA 90:10330, 1993; Altman, J. D. et al., Science 274:94, 1996). Other relatively recent technical developments include staining for intracellular lymphokines, and interferon release assays or ELISPOT assays. Tetramer staining, intracellular lymphokine staining and ELISPOT assays all appear to be at least 10-fold more sensitive than more conventional assays (Lalvani, A. et al., J. Exp. Med. 186:859, 1997; Dunbar, P. R. et al., Curr. Biol. 8:413, 1998; Murali-Krishna, K. et al., Immunity 8:177, 1998).
  • HTL activation may also be assessed using such techniques as T cell proliferation and secretion of lymphokines, e.g. IL-2.
  • Alternatively, immunization of HLA transgenic mice can be used to determine immunogenicity of peptide epitopes. Several transgenic mouse models including mice with human A2.1, A11, and B7 alleles have been characterized and others (e.g., transgenic mice for HLA-A1 and A24) are being developed. HLA-DR1 and HLA-DR3 mouse models have also been developed. Additional transgenic mouse models with other HLA alleles may be generated as necessary. Mice may be immunized with peptides emulsified in Incomplete Freund's Adjuvant and the resulting T cells tested for their capacity to recognize peptide-pulsed target cells and target cells transfected with appropriate genes. CTL responses may be analyzed using cytotoxicity assays described above. Similarly, HTL responses may be analyzed using such assays as T cell proliferation or secretion of lymphokines.
  • IV.G. PREPARATION OF PEPTIDES
  • Peptides in accordance with the invention can be prepared synthetically, by recombinant DNA technology, or from natural sources such as native tumors or pathogenic organisms. Peptide epitopes may be synthesized individually or as polyepitopic peptides. Although the peptide will preferably be substantially free of other naturally occurring host cell proteins and fragments thereof, in some embodiments the peptides may be synthetically conjugated to native fragments or particles.
  • The peptides in accordance with the invention can be a variety of lengths, and either in their neutral (uncharged) forms or in forms which are salts. Peptides may be synthesized The peptides in accordance with the invention are either free of modifications such as glycosylation, side chain oxidation, or phosphorylation; or they contain these modifications, subject to the condition that modifications do not destroy the biological activity of the peptides as described herein.
  • Desirably, the peptide will be as small as possible while still maintaining substantially all of the biological activity of the large peptide. When possible, it may be desirable to optimize HLA class I binding peptides of the invention to a length of about 8 to about 13 amino acid residues, preferably 9 to 10. HLA class II binding peptides may be optimized to a length of about 6 to about 25 amino acids in length, preferably to between about 13 and about 20 residues. Preferably, the peptides are commensurate in size with endogenously processed pathogen-derived peptides or tumor cell peptides that are bound to the relevant HLA molecules. Moreover, the identification and preparation of peptides of other lengths can be carried out using the techniques described herein (e.g., the disclosures regarding primary and secondary anchor positions). However, it is also preferred to identify a larger region of a native peptide that encompasses one and preferably two or more epitopes in accordance with the invention. This sequence is selected on the basis that it contains the greatest number of epitopes per amino acid length. It is to be appreciated that epitopes can be present in a frame-shifted manner, e.g. a 10 amino acid long peptide could contain two 9 amino acid long epitopes and one 10 amino acid long epitope; each epitope can be exposed and bound by an HLA molecule upon administration of a plurality of such peptides. This larger, preferably multi-epitopic, peptide can then be generated synthetically, recombinantly, or via cleavage from the native source.
  • The peptides of the invention can be prepared in a wide variety of ways. For the preferred relatively short size, the peptides can be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart & Young, SOLID PHASE PEPTIDE SYNTHESIS, 2D. ED., Pierce Chemical Co. (1984). Further, individual peptides may be joined using chemical ligation to produce larger peptides.
  • Alternatively, recombinant DNA technology may be employed wherein a nucleotide sequence which encodes an immunogenic peptide of interest is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression. These procedures are generally known in the art, as described generally in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989). Thus, recombinant polypeptides which comprise one or more peptide sequences of the invention can be used to present the appropriate T cell epitope.
  • As the nucleotide coding sequence for peptides of the preferred lengths contemplated herein can be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci, et al., J. Am. Chem. Soc. 103:3185 (1981) modification can be made simply by substituting the appropriate and desired nucleic acid base(s) for those that encode the native peptide sequence. The coding sequence can then be provided with appropriate linkers and ligated into expression vectors commonly available in the art, and the vectors used to transform suitable hosts to produce the desired fusion protein. A number of such vectors and suitable host systems are now available. For expression of the fusion proteins, the coding sequence will be provided with operably linked start and stop codons, promoter and terminator regions and usually a replication system to provide an expression vector for expression in the desired cellular host. For example, promoter sequences compatible with bacterial hosts are provided in plasmids containing convenient restriction sites for insertion of the desired coding sequence. The resulting expression vectors are transformed into suitable bacterial hosts. Of course, yeast, insect or mammalian cell hosts may also be used, employing suitable vectors and control sequences.
  • IV.H. PEPTIDE EPITOPE REAGENTS TO EVALUATE IMMUNE RESPONSES
  • HLA class I and class II binding peptides as described herein can be used, in one embodiment of the invention, as reagents to evaluate an immune response. The immune response to be evaluated may be induced by using as an immunogen any agent that would potentially result in the production of antigen-specific CTLs or HTLs to the peptide epitope(s) to be employed as the reagent. The peptide reagent is not used as the immunogen.
  • For example, a peptide of the invention may be used in a tetramer staining assay to assess peripheral blood mononuclear cells for the presence of antigen-specific CTLs following exposure to a pathogen or immunogen. The HLA-tetrameric complex is used to directly visualize antigen-specific CTLs (see, e.g., Ogg et al. Science 279:2103-2106, 1998; and Altman et al. Science 174:94-96, 1996) and determine the frequency of the antigen-specific CTL population in a sample of peripheral blood mononuclear cells. A tetramer reagent using a peptide of the invention may be generated as follows: A peptide that binds to an allele-specific HLA molecules, or supertype molecules, is refolded in the presence of the corresponding HLA heavy chain and 132-microglobulin to generate a trimolecular complex. The complex is biotinylated at the carboxyl terminal end of the heavy chain at a site that was previously engineered into the protein. Tetramer formation is then induced by the addition of streptavidin. By means of fluorescently labeled streptavidin, the tetramer can be used to stain antigen-specific cells. The cells may then be identified, for example, by flow cytometry. Such an analysis may be used for diagnostic or prognostic purposes.
  • Peptides of the invention may also be used as reagents to evaluate immune recall responses. (see, e.g., Bertoni et al. J. Clin. Invest. 100:503-513, 1997 and Penna et al. J. Exp. Med. 174:1565-1570, 1991.) For example, patient PBC samples from individuals with acute hepatitis B or who have recently recovered from acute hepatitis B may be analyzed for the presence of HBV antigen-specific CTLs using HBV-specific peptides. A blood sample containing mononuclear cells may be evaluated by cultivating the PBCs and stimulating the cells with a peptide of the invention. After an appropriate cultivation period, the expanded cell population may be analyzed for cytotoxic activity.
  • The peptides may also be used as reagents to evaluate the efficacy of a vaccine. PBMCs obtained from a patient vaccinated with an immunogen may be analyzed using, for example, either of the methods described above. A patient is HLA typed, and appropriate peptide reagents that recognize allele-specific molecules present in that patient may be selected for the analysis. The immunogenicity of the vaccine will be indicated by the presence of HBV epitope-specific CTLs in the PBMC sample.
  • IV.I. VACCINE COMPOSITIONS
  • Vaccines that contain as an active ingredient an immunogenically effective amount of one or more peptides as described herein are a further embodiment of the invention. Once appropriately immunogenic epitopes have been defined, they can be sorted and delivered by various means, herein referred to as “vaccine” compositions. Such vaccine compositions can include, for example, lipopeptides (Vitiello, A. et al., J. Clin. Invest. 95:341, 1995), peptides compositions encapsulated in poly(DL-lactide-co-glycolide) (PLG) microspheres (see, e.g., Eldridge, et al. Molec. Immunol. 28:287-294, 1991: Alonso et al. Vaccine 12:299-306, 1994; Jones et al. Vaccine 13:675-681, 1995), peptide compositions encapsulated in immune stimulating complexes (ISCOMS) (see, e.g., Takahashi et al. Nature 344:873-875, 1990; Hu et al. Clin Exp Immunol. 113:235-243, 1998), multiple antigen peptide systems (MAPs) (see e.g., Tam, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988; Tam, J. P., J. Immunol. Methods 196:17-32, 1996), viral delivery vectors (Perkus, M. E. et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 379, 1996; Chakrabarti, S. et al., Nature 320:535, 1986; Hu, S. L. et al., Nature 320:537, 1986; Kieny, M.-P. et al., AIDS Bio/Technology 4:790, 1986; Top, F. H. et al., J. Infect. Dis. 124:148, 1971; Chanda, P. K. et al., Virology 175:535, 1990), particles of viral or synthetic origin (Kofler, N. et al., J. Immunol. Methods. 192:25, 1996; Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993; Falo, L. D., Jr. et al., Nature Med. 7:649, 1995), adjuvants (Warren, H. S., Vogel, F. R., and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; Gupta, R. K. et al., Vaccine 11:293, 1993), liposomes (Reddy, R. et al., J. Immunol. 148:1585, 1992; Rock, K. L., Immunol. Today 17:131, 1996), or, naked or particle absorbed cDNA (Ulmer, J. B. et al., Science 259:1745, 1993; Robinson, H. L., Hunt, L. A., and Webster, R. G., Vaccine 11:957, 1993; Shiver, J. W. et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 423, 1996; Cease, K. B., and Berzofsky, J. A., Annu. Rev. Immunol. 12:923, 1994 and Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993). Toxin-targeted, also know as receptor mediated targeting, delivery technologies also may be used such as those of Avant Immunotherapeutics, Inc. (Needham, Mass.).
  • Furthermore, vaccines in accordance with the invention encompass compositions of one or more of the claimed peptide(s) that can be introduced into a host, including humans, linked to its own carrier, or as a homopolymer or heteropolymer of active peptide units. Such a polymer has the advantage of increased immunological reaction and, where different peptides are used to make up the polymer, the additional ability to induce antibodies and/or CTLs that react with different antigenic determinants of the pathogenic organism or tumor-related peptide targeted for an immune response.
  • Furthermore, useful carriers that can be used with vaccines of the invention are well known in the art, and include, e.g., thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly L-lysine, poly L-glutamic acid, influenza, hepatitis B virus core protein, hepatitis B virus recombinant vaccine and the like. The vaccines can contain a physiologically tolerable (i.e., acceptable) diluent such as water, or saline, preferably phosphate buffered saline. The vaccines also typically include an adjuvant. Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are examples of materials well known in the art. Additionally, as disclosed herein, CTL responses can be primed by conjugating peptides of the invention to lipids, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P3CSS).
  • As disclosed in greater detail herein, upon immunization with a peptide composition in accordance with the invention, via injection, aerosol, oral, transdermal, transmucosal, intrapleural, intrathecal, or other suitable routes, the immune system of the host responds to the vaccine by producing large amounts of CTLs specific for the desired antigen, and the host becomes at least partially immune to later infection, or at least partially resistant to developing an ongoing chronic infection.
  • In some instances it may be desirable to combine the class I peptide vaccines of the invention with vaccines which induce or facilitate neutralizing antibody responses to the target antigen of interest, particularly to viral envelope antigens. A preferred embodiment of such a composition comprises class I and class II epitopes in accordance with the invention. An alternative embodiment of such a composition comprises a class I and/or class II epitope in accordance with the invention, along with a PADRE® (Epimmune, San Diego, Calif.) molecule (described in the related U.S. Ser. No. 08/485,218, which is a CIP of U.S. Ser. No. 08/305,871, now U.S. Pat. No. 5,736,142, which is a CIP of abandoned application U.S. Ser. No. 08/121,101.) Furthermore, any of these embodiments can be administered as a nucleic acid mediated modality.
  • For therapeutic or immunization purposes, the peptides of the invention can also be expressed by viral or bacterial vectors. Examples of expression vectors include attenuated viral hosts, such as vaccinia or fowlpox. This approach involves the use of vaccinia virus as a vector to express nucleotide sequences that encode the peptides of the invention. Upon introduction into an acutely or chronically infected host or into a non-infected host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits a host CTL and/or HTL response. Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover, et al. Nature 351:456-460 (1991). A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention, e.g. adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like, will be apparent to those skilled in the art from the description herein.
  • Antigenic peptides are used to elicit a CTL and/or HTL response ex vivo, as well. The resulting CTL or HTL cells, can be used to treat chronic infections, or tumors in patients that do not respond to other conventional forms of therapy, or will not respond to a therapeutic vaccine peptide or nucleic acid in accordance with the invention. Ex vivo CTL or HTL responses to a particular pathogen (infectious agent or tumor antigen) are induced by incubating in tissue culture the patient's CTL or HTL precursor cells together with a source of antigen-presenting cells (APC), such as dendritic cells, and the appropriate immunogenic peptide. After an appropriate incubation time (typically about 14 weeks), in which the precursor cells are activated, mature and expand into effector cells, the cells are infused back into the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cell (an infected cell or a tumor cell). Transfected dendritic cells may also be used as antigen presenting cells. Alternatively, dendritic cells are transfected, e.g., with a minigene construct in accordance with the invention, in order to elicit immune responses. Minigenes will be discussed in greater detail in a following section.
  • DNA or RNA encoding one or more of the peptides of the invention can also be administered to a patient. This approach is described, for instance, in Wolff et. al., Science 247:1465 (1990) as well as U.S. Pat. Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; WO 98/04720; and in more detail below. Examples of DNA-based delivery technologies include “naked DNA”, facilitated (bupivicaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) delivery.
  • Preferably, the following principles are utilized when selecting an array of epitopes for inclusion in a polyepitopic composition, or for selecting epitopes to be included in a vaccine composition and/or to be encoded by a minigene. It is preferred that each of the following principles are balanced in order to make the selection.
  • 1.) Epitopes are selected which, upon administration, mimic immune responses that have been observed to be correlated with HBV clearance. For HLA Class I this includes 3-4 epitopes that come from at least one antigen of HBV. In other words, it has been observed that in patients who spontaneously clear HBV, that they had generated an immune response to at least 3 epitopes on at least one HBV antigen. For HLA Class II a similar rationale is employed; again 3-4 epitopes are selected from at least one HBV antigen (see e.g., Rosenberg et al. Science 278:1447-1450).
  • 2.) Epitopes are selected that have the requisite binding affinity established to be correlated with immunogenicity: for HLA Class I an IC50 of 500 nM or less, or for Class II an IC50 of 1000 nM or less.
  • 3.) Sufficient supermotif bearing peptides, or a sufficient array of allele-specific motif bearing peptides, are selected to give broad population coverage. For example, it is preferable to have at least 80% population coverage. A Monte Carlo analysis, a statistical evaluation known in the art, can be employed to assess population coverage.
  • 4.) When selecting epitopes from cancer-related antigens it is often preferred to select analogs. When selecting epitopes for infectious disease-related antigens it is often preferable to select native epitopes. Therefore, of particular relevance for infectious disease vaccines (but for cancer-related vaccines as well), are epitopes referred to as “nested epitopes.” Nested epitopes occur where at least two epitopes overlap in a given peptide sequence. A peptide comprising “transcendent nested epitopes” is a peptide that has both HLA class I and HLA class II epitopes in it.
  • When providing nested epitopes, it is preferable to provide a sequence that has the greatest number of epitopes per provided sequence. A limitation on this principle is to avoid providing a peptide that is any longer than the amino terminus of the amino terminal epitope and the carboxyl terminus of the carboxyl terminal epitope in the peptide. When providing a longer peptide sequence, such as a sequence comprising nested epitopes, it is important to screen the sequence in order to insure that it does not have pathological or other deleterious biological properties.
  • 5.) When creating a minigene, as disclosed in greater detail in the following section, an objective is to generate the smallest peptide possible that encompasses the epitopes of interest. The principles employed are similar, if not the same as those employed when selecting a peptide comprising nested epitopes. Thus, upon determination of the nucleic acid sequence to be provided as a minigene, the peptide encoded thereby is analyzed to determine whether any “junctional epitopes” have been created. A junctional epitope is an actual binding epitope, as predicted, e.g., by motif analysis. Junctional epitopes are to be avoided because the recipient may generate an immune response to that epitope. Of particular concern is a junctional epitope that is a “dominant epitope.” A dominant epitope may lead to such a zealous response that immune responses to other epitopes are diminished or suppressed.
  • IV.I.1. Minigene Vaccines
  • A growing body of experimental evidence demonstrates that a number of different approaches are available which allow simultaneous delivery of multiple epitopes. Nucleic acids encoding the peptides of the invention are a particularly useful embodiment of the invention. Epitopes for inclusion in a minigene are preferably selected according to the guidelines above. A preferred means of administering nucleic acids encoding the peptides of the invention uses minigene constructs encoding one or multiple epitopes of the invention. The use of multi-epitope minigenes is described below and in, e.g. An, L. and Whitton, J. L., J. Virol. 71:2292, 1997; Thomson, S. A. et al., J. Immunol. 157:822, 1996; Whitton, J. L. et al., J. Virol. 67:348, 1993; Hanke, R. et al., Vaccine 16:426, 1998. For example, a multi-epitope DNA plasmid encoding nine dominant HLA-A*0201- and A11-restricted epitopes derived from the polymerase, envelope, and core proteins of HBV and HIV, the PADRE® universal helper T cell (HTL) epitope, and an ER-translocating signal sequence was engineered. Immunization of HLA transgenic mice with this plasmid construct resulted in strong CTL induction responses against the nine epitopes tested, similar to those observed with a lipopeptide of known immunogenicity in humans, and significantly greater than immunization in oil-based adjuvants. Moreover, the immunogenicity of DNA-encoded epitopes in vivo correlated with the in vitro responses of specific CTL lines against target cells transfected with the DNA plasmid.
  • For example, to create a DNA sequence encoding the selected epitopes (minigene) for expression in human cells, the amino acid sequences of the epitopes may be reverse translated. A human codon usage table can be used to guide the codon choice for each amino acid. These epitope-encoding DNA sequences may be directly adjoined, so that when translated, a continuous polypeptide sequence is created. To optimize expression and/or immunogenicity, additional elements can be incorporated into the minigene design. Examples of amino acid sequences that could be reverse translated and included in the minigene sequence include: HLA class I epitopes, HLA class II epitopes, a ubiquitination signal sequence, a leader sequence, and/or an endoplasmic reticulum targeting signal. In addition, HLA presentation of CTL and HTL epitopes may be improved by including synthetic (e.g. poly-alanine) or naturally-occurring flanking sequences adjacent to the CTL or HTL epitopes.
  • The minigene sequence may be converted to DNA by assembling oligonucleotides that encode the plus and minus strands of the minigene. Overlapping oligonucleotides (30-100 bases long) may be synthesized, phosphorylated, purified and annealed under appropriate conditions using well known techniques. The ends of the oligonucleotides can be joined, for example, using T4 DNA ligase. This synthetic minigene, encoding the epitope polypeptide, can then be cloned into a desired expression vector.
  • Standard regulatory sequences well known to those of skill in the art are preferably included in the vector to ensure expression in the target cells. Several vector elements are desirable: a promoter with a down-stream cloning site for minigene insertion; a polyadenylation signal for efficient transcription termination; an E. coli origin of replication; and an E. coli selectable marker (e.g. ampicillin or kanamycin resistance). Numerous promoters can be used for this purpose, e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat. Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.
  • Additional vector modifications may be desired to optimize minigene expression and immunogenicity. In some cases, introns are required for efficient gene expression, and one or more synthetic or naturally-occurring introns could be incorporated into the transcribed region of the minigene. The inclusion of mRNA stabilization sequences and sequences for replication in mammalian cells may also be considered for increasing minigene expression.
  • Once an expression vector is selected, the minigene is cloned into the polylinker region downstream of the promoter. This plasmid is transformed into an appropriate E. coli strain, and DNA is prepared using standard techniques. The orientation and DNA sequence of the minigene, as well as all other elements included in the vector, are confirmed using restriction mapping and DNA sequence analysis. Bacterial cells harboring the correct plasmid can be stored as a master cell bank and a working cell bank.
  • In addition, immunostimulatory sequences (ISSs or CpGs) appear to play a role in the immunogenicity of DNA vaccines. These sequences may be included in the vector, outside the minigene coding sequence, if desired to enhance immunogenicity.
  • In some embodiments, a bi-cistronic expression vector which allows production of both the minigene-encoded epitopes and a second protein (included to enhance or decrease immunogenicity) can be used. Examples of proteins or polypeptides that could beneficially enhance the immune response if co-expressed include cytokines (e.g., IL-2, IL-12, GM-CSF), cytokine-inducing molecules (e.g., LeIF) or costimulatory molecules. Helper (HTL) epitopes can be joined to intracellular targeting signals and expressed separately from expressed CTL epitopes; this allows direction of the HTL epitopes to a cell compartment different than that of the CTL epitopes. If required, this could facilitate more efficient entry of HTL epitopes into the HLA class II pathway, thereby improving CTL induction. In contrast to HTL or CTL induction, specifically decreasing the immune response by co-expression of immunosuppressive molecules (e.g. TGF-β) may be beneficial in certain diseases).
  • Therapeutic quantities of plasmid DNA can be produced for example, by fermentation in E. coli, followed by purification. Aliquots from the working cell bank are used to inoculate growth medium, and grown to saturation in shaker flasks or a bioreactor according to well known techniques. Plasmid DNA can be purified using standard bioseparation technologies such as solid phase anion-exchange resins supplied by QIAGEN, Inc. (Valencia, Calif.). If required, supercoiled DNA can be isolated from the open circular and linear forms using gel electrophoresis or other methods.
  • Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is reconstitution of lyophilized DNA in sterile phosphate-buffer saline (PBS). This approach, known as “naked DNA,” is currently being used for intramuscular (IM) administration in clinical trials. To maximize the immunotherapeutic effects of minigene DNA vaccines, an alternative method for formulating purified plasmid DNA may be desirable. A variety of methods have been described, and new techniques may become available. Cationic lipids can also be used in the formulation (see, e.g., as described by WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682 (1988); U.S. Pat. No. 5,279,833; WO 91/06309; and Felgner, et al., Proc. Nat'l Acad. Sci. USA 84:7413 (1987). In addition, glycolipids, fusogenic liposomes, peptides and compounds referred to collectively as protective, interactive, non-condensing (PINC) could also be complexed to purified plasmid DNA to influence variables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types.
  • Target cell sensitization can be used as a functional assay for expression and HLA class I presentation of minigene-encoded CTL epitopes, respectively. For example, the plasmid DNA is introduced into a mammalian cell line that is suitable as a target for standard CTL chromium release assays. The transfection method used will be dependent on the final formulation. Electroporation can be used for “naked” DNA, whereas cationic lipids allow direct in vitro transfection. A plasmid expressing green fluorescent protein (GFP) can be co-transfected to allow enrichment of transfected cells using fluorescence activated cell sorting (FACS). These cells are then chromium-51 (51Cr) labeled and used as target cells for epitope-specific CTL lines; cytolysis, detected by 51Cr release, indicates production of HLA presentation of minigene-encoded CTL epitopes.
  • In vivo immunogenicity is a second approach for functional testing of minigene DNA formulations. Transgenic mice expressing appropriate human HLA proteins are immunized with the DNA product. The dose and route of administration are formulation dependent (e.g., IM for DNA in PBS, IP for lipid-complexed DNA). Twenty-one days after immunization, splenocytes are harvested and restimulated for 1 week in the presence of peptides encoding each epitope being tested. For CTL effector cells, assays are conducted for cytolysis of peptide-loaded, chromium-51 labeled target cells using standard techniques. Lysis of target cells sensitized by HLA loading of peptides corresponding to minigene-encoded epitopes demonstrates DNA vaccine function for in vivo induction of CTLs.
  • Alternatively, the nucleic acids can be administered using ballistic delivery as described, for instance, in U.S. Pat. No. 5,204,253. Using this technique, particles comprised solely of DNA are administered. In a further alternative embodiment, DNA can be adhered to particles, such as gold particles.
  • IV.I.2. Combinations with Helper Peptides
  • The peptides of the present invention, or analogs thereof, which have immunostimulatory activity may be modified to provide desired attributes, such as improved serum half life, or to enhance immunogenicity.
  • For instance, the ability of the peptides to induce CTL activity can be enhanced by linking the peptide to a sequence which contains at least one epitope that is capable of inducing a T helper cell response. Particularly preferred immunogenic peptides/T helper conjugates are linked by a spacer molecule. The spacer is typically comprised of relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions. The spacers are typically selected from, e.g., Ala, Gly, or other neutral spacers of nonpolar amino acids or neutral polar amino acids. It will be understood that the optionally present spacer need not be comprised of the same residues and thus may be a hetero- or homo-oligomer. When present, the spacer will usually be at least one or two residues, more usually three to six residues. Alternatively, the CTL peptide may be linked to the T helper peptide without a spacer.
  • The immunogenic peptide may be linked to the T helper peptide either directly or via a spacer either at the amino or carboxy terminus of the CTL peptide. The amino terminus of either the immunogenic peptide or the T helper peptide may be acylated. The T helper peptides used in the invention can be modified in the same manner as CTL peptides. For instance, they may be modified to include D-amino acids or be conjugated to other molecules such as lipids, proteins, sugars and the like. Exemplary T helper peptides include tetanus toxoid 830-843, influenza 307-319, and malarial circumsporozoite 382-398 and 378-389.
  • In certain embodiments, the T helper peptide is one that is recognized by T helper cells present in the majority of the population. This can be accomplished by selecting amino acid sequences that bind to many, most, or all of the HLA class II molecules. These are known as “loosely HLA-restricted” or “promiscuous” T helper sequences. Examples of amino acid sequences that are promiscuous include sequences from antigens such as tetanus toxoid at positions 830-843 (QYIKANSKFIGITE; SEQ ID NO:2572), Plasmodium falciparum CS protein at positions 378-398 (DIEKKIAKMEKASSVFNVVNS; SEQ ID NO:2573), and Streptococcus 18 kD protein at positions 116 (GAVDSILGGVATYGAA; SEQ ID NO:2574). Other examples include peptides bearing a DR 1-4-7 supermotif.
  • Alternatively, it is possible to prepare synthetic peptides capable of stimulating T helper lymphocytes, in a loosely HLA-restricted fashion, using amino acid sequences not found in nature (see, e.g., PCT publication WO 95/07707). These synthetic compounds called Pan-DR-binding epitopes (e.g., PADRE®, Epimmune, Inc., San Diego, Calif.) are designed on the basis of their binding activity to most HLA-DR (human HLA class II) molecules. For instance, a pan-DR-binding epitope peptide having the formula: aKXVWANTLKAAa, where “X” is either cyclohexylalanine, phenylalanine, or tyrosine, and a is either D-alanine or L-alanine (SEQ ID NO:2575), has been found to bind to most HLA-DR alleles, and to stimulate the response of T helper lymphocytes from most individuals, regardless of their HLA type.
  • T helper epitopes can also be modified to alter their biological properties. For example, peptides presenting T helper epitopes can contain D-amino acids to increase their resistance to proteases and thus extend their serum half-life. Also, the epitope peptides of the invention can be conjugated to other molecules such as lipids, proteins or sugars, or any other synthetic compounds, to increase their biological activity. Specifically, the T helper peptide can be conjugated to one or more palmitic acid chains at either the amino or carboxyl termini.
  • In some embodiments it may be desirable to include in the pharmaceutical compositions of the invention at least one component which primes cytotoxic T lymphocytes. Lipids have been identified as agents capable of priming CTL in vivo against viral antigens. For example, palmitic acid residues can be attached to the ε- and α-amino groups of a lysine residue and then linked, e.g., via one or more linking residues such as Gly, Gly-Gly-, Ser, Ser-Ser, or the like, to an immunogenic peptide. The lipidated peptide can then be administered either directly in a micelle or particle, incorporated into a liposome, or emulsified in an adjuvant, e.g., incomplete Freund's adjuvant. In a preferred embodiment, a particularly effective immunogenic comprises palmitic acid attached to ε- and α-amino groups of Lys, which is attached via linkage, e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.
  • As another example of lipid priming of CTL responses, E. coli lipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P3CSS) can be used to prime virus specific CTL when covalently attached to an appropriate peptide. See, Deres, et al., Nature 342:561 (1989). Peptides of the invention can be coupled to P3CSS, for example, and the lipopeptide administered to an individual to specifically prime a CTL response to the target antigen. Moreover, because the induction of neutralizing antibodies can also be primed with P3CSS-conjugated epitopes, two such compositions can be combined to more effectively elicit both humoral and cell-mediated responses to infection.
  • In addition, additional amino acids can be added to the termini of a peptide to provide for ease of linking peptides one to another, for coupling to a carrier support, or larger peptide, for modifying the physical or chemical properties of the peptide or oligopeptide, or the like. Amino acids such as tyrosine, cysteine, lysine, glutamic or aspartic acid, or the like, can be introduced at the C- or N-terminus of the peptide or oligopeptide, particularly class I peptides. However, it is to be noted that modification at the carboxyl terminus may, in some cases, alter binding characteristics of the peptide. In addition, the peptide or oligopeptide sequences can differ from the natural sequence by being modified by terminal-NH2 acylation, e.g., by alkanoyl (C1-C20) or thioglycolyl acetylation, terminal-carboxyl amidation, e.g., ammonia, methylamine, etc. In some instances these modifications may provide sites for linking to a support or other molecule.
  • IV.J. ADMINISTRATION OF VACCINES FOR THERAPEUTIC OR PROPHYLACTIC PURPOSES
  • The peptides of the present invention and pharmaceutical and vaccine compositions of the invention are useful for administration to mammals, particularly humans, to treat and/or prevent HBV infection. Vaccine compositions containing the peptides of the invention are administered to a patient susceptible to or otherwise at risk for HBV infection to elicit an immune response against HBV antigens and thus enhance the patient's own immune response capabilities. In therapeutic applications, compositions are administered to a patient in an amount sufficient to elicit an effective CTL response to the virus or tumor antigen and to cure or at least partially arrest or slow symptoms and/or complications. An amount adequate to accomplish this is defined as “therapeutically effective dose.” Amounts effective for this use will depend on, e.g., the particular composition administered, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician. Generally the dosage range for an initial immunization (i.e., therapeutic or prophylactic administration) is between about 1.0 μg to about 5000 μg of peptide, typically between about 10 μg to about 1000 mg, for a 70 kg patient, followed by boosting dosages of between about 1.0 μg to about 5000 μg of peptide pursuant to a boosting regimen over weeks to months depending upon the patient's response and condition as determined by measuring specific CTL activity in the patient's blood. The peptides and compositions of the present invention may be employed in serious disease states, that is, life-threatening or potentially life threatening situations. In such cases, as a result of the minimal amounts of extraneous substances and the relative nontoxic nature of the peptides in preferred compositions of the invention, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these peptide compositions relative to these stated dosage amounts.
  • As noted above, the “CTL” peptides of the invention induce immune responses when contacted with a CTL specific to an epitope comprised by the peptide. The manner in which the peptide is contacted with the CTL is not critical to the invention. For instance, the peptide can be contacted with the CTL either in vivo or in vitro. If the contacting occurs in vivo, the peptide itself can be administered to the patient, or other vehicles, e.g., DNA vectors encoding one or more peptides, viral vectors encoding the peptide(s), liposomes and the like, can be used, as described herein.
  • For pharmaceutical compositions, the immunogenic peptides, or DNA encoding them, are generally administered to an individual already infected with HBV. The peptides or DNA encoding them can be administered individually or as fusions of one or more peptide sequences. Those in the incubation phase or the acute phase of infection can be treated with the immunogenic peptides separately or in conjunction with other treatments, as appropriate.
  • For therapeutic use, administration should generally begin at the first diagnosis of HBV infection. This is followed by boosting doses until at least symptoms are substantially abated and for a period thereafter. In chronic infection, loading doses followed by boosting doses may be required.
  • Treatment of an infected individual with the compositions of the invention may hasten resolution of the infection in acutely infected individuals. For those individuals susceptible (or predisposed) to developing chronic infection, the compositions are particularly useful in methods for preventing the evolution from acute to chronic infection. Where susceptible individuals are identified prior to or during infection, the composition can be targeted to them, minimizing need for administration to a larger population.
  • The peptide or other compositions as used for the treatment of chronic HBV infection and to stimulate the immune system to eliminate pathogen-infected cells in, e.g., persons who have not manifested symptoms of disease but who act as a disease vector. In this context, it is generally important to provide an amount of immuno-potentiating peptide in a formulation and mode of administration sufficient to effectively stimulate a cytotoxic T cell response; compositions which stimulate helper T cell responses can also be given in accordance with this embodiment of the invention. Thus, for treatment of chronic infection, a representative dose is in the range of about 1.0 μg to about 5000 μg, preferably about 10 μg to 1000 μg, per 70 kg patient weight per dose. Immunizing doses followed by boosting doses at established intervals, e.g., from four weeks to six months, may be required, possibly for a prolonged period of time to effectively immunize an individual. In the case of chronic infection, administration should continue until at least clinical symptoms or laboratory tests indicate that the viral infection has been eliminated or substantially abated and for a period thereafter. The dosages, routes of administration, and dose schedules are adjusted in accordance with methodologies known in the art.
  • The pharmaceutical compositions for therapeutic treatment are intended for parenteral, topical, oral, intrathecal, or local administration. Preferably, the pharmaceutical compositions are administered parentally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. Thus, the invention provides compositions for parenteral administration which comprise a solution of the immunogenic peptides dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used, e.g., water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • The concentration of peptides of the invention in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
  • The peptides of the invention may also be administered via liposomes, which serve to target the peptides to a particular tissue, such as lymphoid tissue, or targeted selectively to infected cells, as well as increase the half-life of the peptide composition. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations the peptide to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to, e.g., a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions. Thus, liposomes either filled or decorated with a desired peptide of the invention can be directed to the site of lymphoid cells, where the liposomes then deliver the peptide compositions. Liposomes for use in the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka, et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.
  • For targeting cells of the immune system, a ligand to be incorporated into the liposome can include, e.g., antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells. A liposome suspension containing a peptide may be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the manner of administration, the peptide being delivered, and the stage of the disease being treated.
  • For solid compositions, conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more peptides of the invention, and more preferably at a concentration of 25%-75%.
  • For aerosol administration, the immunogenic peptides are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of peptides are 0.01%-20% by weight, preferably 1%-10%. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute 0.1%-20% by weight of the composition, preferably 0.25-5%. The balance of the composition is ordinarily propellant. A carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery.
  • The vaccine compositions of the invention may also be used purely as prophylactic agents. Vaccine compositions containing the peptide epitopes of the invention are administered to a patient susceptible to, or otherwise at risk for, HBV infection to elicit an immune response against HBV antigens and thus enhance the patient's own immune response capabilities following exposure to HBV. Generally the dosage range for an initial prophylactic immunization is between about 1.0 μg to about 5000 μg of peptide, typically between about 10 μg to about 1000 μg, for a 70 kg patient. This is followed by boosting dosages of between about 1.0 μg to about 5000 μg of peptide administered at defined intervals from about four weeks to six months after the initial administration of vaccine. The immunogenicity of the vaccine may be assessed by measuring specific CTL activity in the patient's blood.
  • IV.K. KITS
  • The peptide and nucleic acid compositions of this invention can be provided in kit form together with instructions for vaccine administration. Typically the kit would include desired peptide compositions in a container, preferably in unit dosage form and instructions for administration. An alternative kit would include a minigene construct with desired nucleic acids of the invention in a container, preferably in unit dosage form together with instruction for administration. Lymphokines such as IL-2 or IL-12 may also be included in the kit. Other kit components that may also be desirable include, for example, a sterile syringe, booster dosages, and other desired excipients.
  • The invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of non-critical parameters that can be changed or modified to yield alternative embodiments in accordance with the invention.
  • V. EXAMPLES Example 1 HLA Class I Binding Assays
  • The following example of peptide binding to HLA-A3 supertype molecules demonstrates quantification of binding affinities of HLA class I peptides. Analogous binding assays can be performed for other peptides that bind class I or class II HLA molecules. Furthermore, binding assays can be performed with peptides that are not motif-bearing.
  • For example, the affinity of peptides bearing an HLA-A3 supermotif was determined as follows. Epstein-Barr virus (EBV)-transformed homozygous cell lines were used as sources of class I molecules. Cell lines include, e.g., GM3107 (A3, B7; Human Genetic Mutant Repository); BVR (A11, B35.3, Cw4; Human Genetic Mutant Repository); SPACH (A31, B62, Cw1/3; ASHI Repository Collection); LWAGS (A*3301, B14, and Cw8; ASHI Repository Collection) (Bodmer, et al., Hum. Immunol. 43:149, 1995), and a C1R transfectant characterized by Dr. Walter Storkus (University of Pittsburgh) for the isolation of A*6801. Cell lines were maintained as previously described (Sidney, et al., J. Immunol. 154:247 (1995); Sette, et al., Mol. Immunol. 31:813 (1994)).
  • Cell lysates were prepared and HLA class I molecules purified in accordance with disclosed protocols (Sidney, et al., J. Immunol. 154:247 (1995); Sette, et al., Mol. Immunol. 31:813 (1994)). Briefly, cells were lysed at a concentration of 108 cells/ml in 50 mM Tris-HCl, pH 8.5, containing 1% Nonidet P-40 (Fluka Biochemika, Buchs, Switzerland), 150 mM NaCl, 5 mM EDTA, and 2 mM PMSF. The lysates were passed through 0.45 μM filters and cleared of nuclei and debris by centrifugation at 10,000 g for 20 minutes. HLA proteins were then purified by affinity chromatography. Columns of inactivated Sepharose CL 4B and Protein A Sepharose were used as precolumns. The cell lysate was depleted of HLA-B and HLA-C proteins by repeated passage over Protein A Sepharose beads conjugated with the anti-HLA(B,C) antibody B1.23.2 (Rebai, et al., Tissue Antigens 22:107 (1983)). Typically two to four passages were required for effective depletion. Subsequently, the anti HLA(A,B,C) antibody W6/32 (Barnstable, et al., Cell 14:9 (1978)) was used to capture HLA-A molecules. Protein purity, concentration, and effectiveness of depletion steps were monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
  • Binding Assays
  • Quantitative assays for the binding of peptides to soluble class I molecules on the basis of the inhibition of binding of a radiolabeled standard probe peptide to detergent solubilized HLA molecules were performed as described in the literature (Kubo, et al., J. Immunol. 152:3913 (1994); Kast, et al., J. Immunol. 152:3904 (1994); Sidney, et al., J. Immunol. 154:247 (1995); Sette, et al., Mol. Immunol. 31:813 (1994); Ruppert, et al., Cell 74:929 (1993)). Briefly, 1-10 nM of radiolabeled probe peptide, iodinated by the Chloramine-T method (Greenwood, et al., Biochem. J. 89:114 (1963)), was co-incubated at room temperature with various amounts of HLA in the presence of 1 μM human β2-microglobulin (Scripps Laboratories, San Diego, Calif., USA) and a cocktail of protease inhibitors. At the end of a two day incubation period, the percent of HLA-bound radioactivity was determined by size exclusion gel filtration chromatography on a TSK 2000 column.
  • The A3CON1 peptide (sequence KVFPYALINK; SEQ ID NO:2576) (Kubo, et al., J. Immunol. 152:3913 (1994)) was used as the radiolabeled probe for the A3, A11, A31, and A*6801 assays. A T7Y analogue of HBVc 141-151 (sequence STLPETYVVRR; SEQ ID NO:2577) (Missale, et al., J. Exp. Med. 177:751 (1993)) was used as the radiolabeled probe for the A*3301 assay. In the case of competitive assays, the concentration of peptide yielding 50% inhibition of the binding of the radiolabeled probe peptide (IC50) was calculated. Peptides were usually tested at one or two high doses, and the IC50 of peptides yielding positive inhibition were determined in subsequent experiments, in which two to six further dilutions were tested, as necessary. To achieve a suitable signal, HLA concentrations yielding approximately 15% binding of the radiolabeled probe peptide were used for all competitive inhibition assays. Under these conditions the concentration of the labeled peptide is less than the concentration of the HLA molecule and the IC50 is less than the concentration of the HLA molecule, therefore the measured IC50s are reasonable approximations of the true KD values. Each competitor peptide was tested in two to four completely independent experiments. As a positive control, in each experiment, the unlabeled version of the relevant radiolabeled probe was tested and its IC50 measured. The average IC50 of A3CON1 for the A3, A11, A31, and A*6801 assays were 11, 6, 18, and 8 nM, respectively. The average IC50 of the HBVc 141-151 peptide in the A*3301 assay was 29 nM.
  • Example 2 Implementation of the Extended Supermotif to Improve the Binding Capacity of Native Peptides by Creating Analogs
  • HLA motifs and supermotifs (comprising primary and/or secondary residues) are useful in preparing highly cross-reactive native peptides, as demonstrated herein. Moreover, the definition of HLA motifs and supermotifs also allows one to engineer highly cross-reactive epitopes by identifying residues within a native peptide sequence which can be analoged, or “fixed”, to confer upon a peptide certain characteristics, e.g., greater cross-reactivity within the group of HLA molecules that make-up the supertype, and/or greater binding affinity for some or all of those HLA molecules Examples of analog peptides that exhibit modulated binding affinity are provided.
  • Analogs representing primary anchor single amino acid residues substituted with I residues at the C-terminus of two different B7-like peptides (HBV env 313 and HBV pol 541) were synthesized and tested for their B7-supertype binding capacity. It was found that the I substitution had an overall positive effect on binding affinity and/or cross-reactivity in both cases. In the case of HBV env 313 the 19 (I at C-terminal position 9) replacement was effective in increasing cross-reactivity from 4 to 5 alleles bound by virtue of an almost 400-fold increase B*5401 binding affinity. In the case of HBV pol 541, increased cross-reactivity was similarly achieved by a substantial increase in B*5401 binding. Also, significant gains in binding affinity for B*0702, B51, and B*5301 were observed with the HBV pol 541 I9 analog.
  • Moreover, HLA supermotifs are of value in engineering highly cross-reactive peptides by identifying particular residues at secondary anchor positions that are associated with such cross-reactive properties. Demonstrating this, the capacity of a second set of peptides representing discreet single amino acid substitutions at positions one and three of five different B7-supertype binding peptides were synthesized and tested for their B-7 supertype binding capacity. In 4/4 cases the effect of replacing the native residue at position 1 with the aromatic residue F (an “F1” substitution) resulted in an increase in cross-reactivity, compared to the parent peptide, and, in most instances, binding affinity was increased three-fold or better (Table XXII). More specifically, for HBV env 313, MAGE2 170, and HCV core 168 complete supertype cross-reactivity was achieved with the F1 substitution analogs. These gains were achieved by dramatically increasing B*5401 binding affinity. Also, gains in affinity were noted for other alleles in the cases of HCV core 168 (B*3501 and B*5301) and MAGE2 170 (B*3501, B51 and B*5301). Finally, in the case of MAGE3 196, the F1 replacement was effective in increasing cross-reactivity because of gains in B*0702 binding. An almost 70-fold increase in B51 binding capacity was also noted.
  • Two analogs were also made using the supermotif positive F substitution at position three (an “F3” substitution). In both instances increases in binding affinity and cross-reactivity were achieved. Specifically, in the case of HBV pol 541, the F3 substitution was effective in increasing cross-reactivity by virtue of its effect on B*5401 binding. In the case of MAGE3 196, complete supertype cross-reactivity was achieved by increasing B*0702 and B*3501 binding capacity. Also, in the case of MAGE3 196, it is notable that increases in binding capacity between 40- and 5000-fold were obtained for B*3501, B51, B*5301, and B*5401.
  • In conclusion, these data demonstrate that by the use of even single amino acid substitutions, it is possible to increase the binding affinity and/or cross-reactivity of peptide ligands for HLA supertype molecules.
  • Example 3 Induction of HLA-Restricted CTL by Subcutaneous Priming With HBV Peptide in Incomplete Freund's Adjuvant (IFA)
  • The immunogenicity of HLA class I binding peptides can be assessed in vivo as described in, e.g., Sette et al. J. Immunol. 153:5586-5592 (1994). This example illustrates such a procedure, whereby subcutaneous injection of HBV peptide in Incomplete Freund's Adjuvant (IFA) can be used to induce HBV-specific CTL in mice that are transgenic for a human HLA allele such as the human HLA-A11 allele.
  • Priming and In Vitro Restimulation: Mice that are transgenic for HLA-A11, (e.g. HLA-A11/Kb strain) are injected with 100 microliters of an emulsion of purified HBV peptide in IFA. The purified peptide comprises an A11 motif, and is selected from the preferred peptides listed in Table XVI or, alternatively, may be an analog peptide. The peptide epitope (50 μg/mouse) and equimolar amounts of the helper epitope HBV core 128-140 (140 μg/mouse) are dissolved in PBS/5% DMSO, emulsified in WA, and injected subcutaneously at the base of the tail of the transgenic mice. Eleven days after priming, splenocytes (5×106 cells/well in a 24-well plate) obtained from these animals are restimulated with syngeneic irradiated LPS blasts (2×106/well) coated with peptide.
  • LPS blasts from unprimed HLA-A11 transgenic mice are prepared 72 hours before use by suspending splenocytes in medium containing LPS (25 μg/ml) and dextran sulfate (7 μg/ml). Coating is achieved by incubating 50 μg of peptide with 1.2×106 LPS blasts in a volume of 0.4 ml of RPMI medium supplemented with 10% FCS for 1 hour at 37° C. The cells are washed once and then co-cultured with splenocytes. After six days, effector cells are assayed, as outlined for example in Example 5, for cytotoxicity against 51Cr-labeled 3A4-721.221-A11/Kb target cells in the presence of the peptide.
  • The effector cells (2×106 cells/well) are re-stimulated at weekly intervals. For the first re-stimulation, peptide-coated LPS blasts are used, followed by peptide-coated A11/Kb cells. Six days after re-stimulation, effector cells are assayed for cytotoxicity as above.
  • Example 4 Recognition of Generation of Endogenous Processed Antigens After Priming
  • This example determines that CTL induced by in vivo priming with peptide (as disclosed in Example 3) recognize endogenously synthesized antigens.
  • Effector cells from the procedure disclosed in Example 3 are re-stimulated in vitro using peptide-coated stimulator cells. Six days later, effector cells are assayed for cytotoxicity and the cell lines that contain peptide-specific cytotoxic activity are further re-stimulated. An additional six days later, these cell lines are tested for cytotoxic activity on 51Cr labeled 3A4-721.221-A11/Kb target cells, in the absence or presence of peptide, and also tested on 51Cr labeled target cells bearing the endogenously synthesized antigen.
  • The result will demonstrate that CTL lines obtained from animals primed with peptide epitope recognize endogenously synthesized HBV antigen.
  • Example 5 Activity of CTL-HTL Conjugated Epitopes in Transgenic Mice
  • This example illustrates the induction of CTLs in transgenic mice by use of an HBV CTL/HTL peptide conjugate. An analogous study may be found in Oseroff et al. Vaccine 16:823-833 (1998). The peptide composition can comprise multiple CTL and/or HTL epitopes. Such a peptide composition can comprise a lipidated HTL epitope conjugated to a preferred CTL epitope containing, for example, an A11 motif or an analog of that epitope.
  • Lipopeptides are prepared by coupling the appropriate fatty acid to the amino terminus of the resin bound peptide. A typical procedure is as follows: A dichloromethane solution of a four-fold excess of a pre-formed symmetrical anhydride of the appropriate fatty acid is added to the resin and the mixture is allowed to react for two hours. The resin is washed with dichloromethane and dried. The resin is then treated with trifluoroacetic acid in the presence of appropriate scavengers [e.g. 5% (v/v) water] for 60 minutes at 20° C. After evaporation of excess trifluoroacetic acid, the crude peptide is washed with diethyl ether, dissolved in methanol and precipitated by the addition of water. The peptide is collected by filtration and dried.
  • Preparation of Peptides for Immunization: Peptide Compositions are Typically resuspended in DMSO at a concentration of 20 mg/ml. Before use, peptides are prepared at the required concentration by dilution in saline or the appropriate medium.
  • Immunization procedures: A11/Kb mice, which are transgenic for the human HLA A11 allele, are primed subcutaneously (base of the tail) with 0.1 ml of peptide conjugate formulated in saline, or DMSO/saline. Seven days after priming, splenocytes obtained from these animals are restimulated with syngeneic irradiated LPS-activated lymphoblasts coated with peptide.
  • Media:
  • a. RPMI-1640 supplemented with 10% fetal calf serum (FCS) 2 mM Glutamine, 50 μg/ml Gentamicin and 5×10−5 M 2-mercaptoethanol serves as culture medium
  • b. RPMI-1640 containing 25 mM HEPES buffer and supplemented with 2% (FCS) is used as cell washing medium.
  • Cell lines: The 3A4-721.221-A11/Kb cell line is used as target cells. This cell line is an EBV transformed cell line that was mutagenized and selected to be Class I negative which was transfected with an HLA-A11/Kb gene.
  • LPS-activated lymphoblasts: Splenocytes obtained from transgenic mice are resuspended at a concentration of 1-1.5×106/m1 in culture medium supplemented with 25 μg/ml LPS and 7 μg/ml dextran sulfate in 75 cm2 tissue culture flasks. After 72 hours at 37° C., the lymphoblasts are collected for use by centrifugation.
  • Peptide coating of lymphoblasts: Peptide coating of the LPS activated lymphoblasts is achieved by incubating 30×106 irradiated (3000 rads) lymphoblasts with 100 μg of peptide in 1 ml of R10 medium for 1 hr at 37° C. Cells are then washed once and resuspended in culture medium at the desired concentration.
  • In vitro CTL activation: One week after priming, spleen cells (30×106 cells/flask) are co-cultured at 37° C. with syngeneic, irradiated (3000 rads), peptide coated lymphoblasts (10×106 cells/flask) in 10 ml of culture medium/T25 flask. After six days, the effector cells are harvested and assayed for cytotoxic activity.
  • Assay for cytotoxic activity: Target cells (1.0-1.5×106) are incubated at 37° C. in the presence of 200 μl of sodium 51Cr chromate. After 60 minutes, cells are washed three times and resuspended in R10 medium. Peptide is added where required at a concentration of 1 μg/ml. For the assay, 104 51Cr-labeled target cells are added to different concentrations of effector cells (final volume of 200 μl) in U-bottom 96-well plates. After a 6 hour incubation period at 37° C., a 0.1 ml aliquot of supernatant is removed from each well and radioactivity is determined in a Micromedic automatic gamma counter. The percent specific lysis is determined by the formula: percent specific release=100×(experimental release−spontaneous release)/(maximum release−spontaneous release). To facilitate comparison between separate CTL assays run under the same conditions, % 51Cr release data is expressed as lytic units/106 cells. One lytic unit is arbitrarily defined as the number of effector cells required to achieve 30% lysis of 10,000 target cells in a 6 hour 51Cr release assay. To obtain specific lytic units/106, the lytic units/106 obtained in the absence of peptide is subtracted from the lytic units/106 obtained in the presence of peptide. For example, if 30% 51Cr release is obtained at the E:T of 50:1 (i.e., 5×105 effector cells for 10,000 targets) in the absence of peptide and 5:1 (i.e., 5×104 effector cells for 10,000 targets) in the presence of peptide, the specific lytic units would be: (1×106(5×104)−(1×106(5×105)=18 LU/106.
  • The results are analyzed to assess the magnitude of the CTL responses of animals injected with the immunogenic CTL/HTL conjugate vaccine preparation. Analyses similar to this may be performed to evaluate the immunogenicity of peptide conjugates containing multiple CTL epitopes and/or multiple HTL epitopes. In accordance with these procedures it is found that CTL and HTL responses are induced.
  • Example 7 Induction of Specific CTL Response in Humans
  • A human clinical trial for an immunogenic composition comprising CTL and HTL epitopes is set up as an IND Phase I, dose escalation study (5, 50 and 500 μg) and carried out as a randomized, double-blind, placebo-controlled trial. Such a trial is designed, for example, as follows:
  • A total of about 27 subjects are enrolled and divided into 3 groups:
  • Group I: 3 subjects are injected with placebo and 6 subjects are injected with 5 μg of peptide composition;
  • Group II: 3 subjects are injected with placebo and 6 subjects are injected with 50 μg peptide composition;
  • Group III: 3 subjects are injected with placebo and 6 subjects are injected with 500 μg of peptide composition.
  • After 4 weeks following the first injection, all subjects receive a booster inoculation at the same dosage.
  • The endpoints measured in this study relate to the safety and tolerability of the peptide composition as well as its immunogenicity. Cellular immune responses to the peptide composition are an index of the intrinsic activity of this the peptide composition, and can therefore be viewed as a measure of biological efficacy. The following summarize the clinical and laboratory data that relate to safety and efficacy endpoints.
  • Safety: The incidence of adverse events is monitored in the placebo and drug treatment group and assessed in terms of degree and reversibility.
  • Evaluation of Vaccine Efficacy: For evaluation of vaccine efficacy, subjects are bled before and after injection. Peripheral blood mononuclear cells are isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation, aliquoted in freezing media and stored frozen. Samples are assayed for CTL and HTL activity.
  • Thus, the vaccine is found to be both safe and efficacious.
  • Example 8 Phase II Trials in Patients Infected with HBV
  • Phase II trials are performed to study the effect of administering the CTL-HTL peptide compositions to patients (male and female) having chronic HBV infection. A main objective of the trials is to determine an effective dose and regimen for inducing CTLs in chronically infected HBV patients, to establish the safety of inducing a CTL response in these patients, and to see to what extent activation of CTLs improves the clinical picture of chronically infected CTL patients, as manifested by a transient flare in alanine aminotransferase (ALT), normalization of ALT, and reduction in HBV DNA. Such a study is designed, for example, as follows:
  • The studies are performed in multiple centers in the U.S. and Canada. The trial design is an open-label, uncontrolled, dose escalation protocol wherein the peptide composition is administered as a single dose followed six weeks later by a single booster shot of the same dose. The dosages are 50, 500 and 5,000 micrograms per injection. Drug-associated adverse effects are recorded.
  • There are three patient groupings. The first group is injected with 50 micrograms of the peptide composition and the second and third groups with 500 and 5,000 micrograms of peptide composition, respectively. The patients within each group range in age from 21-65 and include both males and females. The patients represent diverse ethnic backgrounds. All of them are infected with HBV for over five years and are HIV, HCV and HDV negative, but have positive levels of HBe antigen and HBs antigen.
  • The magnitude and incidence of ALT flares and the levels of HBV DNA in the blood are monitored to assess the effects of administering the peptide compositions. The levels of HBV DNA in the blood are an indirect indication of the progress of treatment. The vaccine composition is found to be both safe and efficacious in the treatment of chronic HBV infection.
  • Example 9 Selection of CTL and HTL Epitopes for Inclusion in an HBV-Specific Vaccine
  • This example illustrates the procedure for the selection of peptide epitopes for vaccine compositions of the invention.
  • The following principles are utilized when selecting an array of epitopes for inclusion in a polyepitopic composition, or for selecting epitopes to be included in a vaccine composition and/or to be encoded by a minigene. Each of the following principles are balanced in order to make the selection.
  • 1.) Epitopes are selected which, upon administration, mimic immune responses that have been observed to be correlated with HBV clearance. For HLA Class I this includes 3-4 epitopes that come from at least one antigen of HBV. In other words, it has been observed that in patients who spontaneously clear HBV, that they had generated an immune response to at least 3 epitopes on at least one HBV antigen. For HLA Class II a similar rationale is employed; again 3-4 epitopes are selected from at least one HBV antigen.
  • 2.) Epitopes are selected that have the requisite binding affinity established to be correlated with immunogenicity: for HLA Class I an IC50 of 500 nM or less, or for Class II an IC50 of 1000 nM or less.
  • 3.) Sufficient supermotif bearing peptides, or a sufficient array of allele-specific motif bearing peptides, are selected to give broad population coverage. For example, epitopes are selected to provide at least 80% population coverage. A Monte Carlo analysis, a statistical evaluation known in the art, is employed to assess population coverage.
  • 4.) When selecting epitopes for HBV antigens it is often preferable to select native epitopes. Therefore, of particular relevance for infectious disease vaccines, are epitopes referred to as “nested epitopes.” Nested epitopes occur where at least two epitopes overlap in a given peptide sequence. A peptide comprising “transcendent nested epitopes” is a peptide that has both HLA class I and HLA class II epitopes in it.
  • When providing nested epitopes, a sequence that has the greatest number of epitopes per provided sequence is provided. A limitation on this principle is to avoid providing a peptide that is any longer than the amino terminus of the amino terminal epitope and the carboxyl terminus of the carboxyl terminal epitope in the peptide. When providing a longer peptide sequence, such as a sequence comprising nested epitopes, the sequence is screened in order to insure that it does not have pathological or other deleterious biological properties.
  • 5.) When creating a minigene, as disclosed in greater detail in the Example 10, an objective is to generate the smallest peptide possible that encompasses the epitopes of interest. The principles employed are similar, if not the same as those employed when selecting a peptide comprising nested epitopes. Thus, upon determination of the nucleic acid sequence to be provided as a minigene, the peptide encoded thereby is analyzed to determine whether any “junctional epitopes” have been created. A junctional epitope is an actual binding epitope, as predicted, e.g., by motif analysis. Junctional epitopes are to be avoided because the recipient may generate an immune response to that epitope. Of particular concern is a junctional epitope that is a “dominant epitope.” A dominant epitope may lead to such a zealous response that immune responses to other epitopes are diminished or suppressed.
  • Peptide epitopes for inclusion in vaccine compositions are, for example, selected from those listed in Table XXIII. A vaccine composition comprised of selected peptides, when administered, is safe, efficacious, and elicits an immune response similar in magnitude of an immune response that clears an acute HBV infection.
  • Example 10 Construction of Minigene Multi-Epitope DNA Plasmids
  • Expression plasmids have been constructed and evaluated as described, for example, in U.S. Ser. No. 60/085,751 filed May 15, 1998 and U.S. Ser. No. 09/078,904 filed May 13, 1998. The binding peptide epitopes and their positions for some of the plasmids described therein are shown in FIG. 1 as example of the orientation of peptide epitopes in minigene constructs. Such a plasmid may, for example, also include multiple CTL and HTL peptide epitopes. In the present example, HLA-A11 motif-bearing peptides are used in conjunction with DR supermotif-bearing peptides. Preferred A11 epitopes are identified, for example, in Table XVI or Table XXI and peptide epitopes recognized by HLA DR molecules (Tables XVIII and XIX). Four class I A11 motif-bearing peptide epitopes or analogs of those peptide epitopes derived from the same HBV antigen, e.g. the envelope protein, are selected as CTL epitopes. Four class II motif-bearing peptide epitopes derived from the same antigen, e.g., the envelope protein, are selected as HTL epitopes. These epitopes are then incorporated into a minigene for expression in an expression vector.
  • This example illustrates the methods to be used for construction of such a minigene-bearing expression plasmid. Other expression vectors that may be used for minigene compositions are available and known to those of skill in the art.
  • A pMin minigene DNA plasmid is constructed from an early generation DNA plasmid designated as pMin.0. This plasmid contains a consensus Kozak sequence and a consensus murine kappa Ig-light chain signal sequence followed by a string of CTL and HTL epitopes selected in accordance with principles disclosed herein. The pMIN sequence encodes an open reading frame fused to the Myc and His antibody epitope tag coded for by the pcDNA 3.1 Myc-His vector.
  • Overlapping oligonucleotides, for example eight oligonucleotides, averaging approximately 70 nucleotides in length with 15 nucleotide overlaps, are synthesized and HPLC-purified. The oligonucleotides encode the selected peptide epitopes as well as appropriate linker nucleotides. The final multiepitope minigene is assembled by extending the overlapping oligonucleotides in three sets of reactions using PCR. A Perkin/Elmer 9600 PCR machine is used and a total of 30 cycles are performed using the following conditions: 95° C. for 15 sec, annealing temperature (5° below the lowest calculated Tm of each primer pair) for 30 sec, and 72° C. for 1 min.
  • For the first PCR reaction, 5 μg of each of two oligonucleotides are annealed and extended: Oligonucleotides 1+2, 3+4, 5+6, and 7+8 are combined in 100 μl reactions containing Pfu polymerase buffer (1×=10 mM KCL, 10 mM (NH4)2SO4, 20 mM Tris-chloride, pH 8.75, 2 mM MgSO4, 0.1% Triton X-100, 100 μg/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu polymerase. The full-length dimer products are gel-purified, and two reactions containing the product of 1+2 and 3+4, and the product of 5+6 and 7+8 are mixed, annealed, and extended for 10 cycles. Half of the two reactions are then mixed, and 5 cycles of annealing and extension carried out before flanking primers are added to amplify the full length product for 25 additional cycles. The full-length product is gel-purified and cloned into pCR-blunt (Invitrogen) and individual clones are screened by sequencing.
  • Example 11 The Plasmid Construct and the Degree to which it Induces Immunogenicity
  • The degree to which the plasmid construct prepared using the methodology outlined in Example 10 is able to induce immunogenicity is evaluated through in vivo injections into mice and in vitro CTL culture and cytotoxicity assays as detailed e.g., in U.S. Ser. No. 60/085,751 filed May 15, 1998. To assess the capacity of the pMin minigene construct to induce CTLs in vivo, HLA-A11/Kb transgenic mice are immunized intramuscularly with 100 μg of naked cDNA. As a means of comparing the level of CTLs induced by cDNA immunization, a control group of animals is also immunized with an actual peptide composition that comprises multiple epitopes synthesized as a single polypeptide.
  • Splenocytes from immunized animals are stimulated twice with each of the peptide epitopes encoded in the minigene, then assayed for peptide-specific cytotoxic activity in a 51Cr release assay. The results indicate the magnitude of the CTL response directed against each of its A11-restricted epitopes, thus indicating the in vivo immunogenicity of the minigene vaccine. It is, therefore, found that the minigene elicits immune responses directed toward A11-restricted epitopes.
  • Example 12 Peptide Composition for Prophylactic Uses
  • Vaccine compositions of the present invention are used to prevent HBV infection in persons who are at risk. For example, a polyepitopic peptide epitope composition containing multiple CTL and HTL epitopes such as those selected in Examples 9 and/or 10, which are also selected to target greater than 80% of the population, is administered to individuals at risk for HBV infection. The composition is provided as a single lipidated polypeptide that encompasses multiple epitopes. The vaccine is administered in an aqueous carrier comprised of Freunds Incomplete Adjuvant. The dose of peptide for the initial immunization is from about 1 to about 5,000 μg for a 70 kg patient. The initial administration of vaccine is followed by booster dosages at 4 weeks followed by evaluation of the magnitude of the immune response in the patient by techniques that determine the presence of epitope-specific CTL populations in a PBMC sample. Additional booster doses are administered as required. The composition is found to be both safe and efficacious as a prophylaxis against HBV infection.
  • Alternatively, the polyepitopic peptide composition can be administered as a nucleic acid in accordance with methodologies known in the art and disclosed herein.
  • Example 13 Polyepitopic Vaccine Compositions Derived from Native HBV Sequences
  • A native HBV polyprotein sequence is screened, preferably using computer algorithms defined for each class I and/or class II supermotif or motif, to identify “relatively short” regions of the polyprotein that comprise multiple epitopes. This relatively short sequence that contains multiple distinct, even overlapping, epitopes is selected and used to generate a minigene construct. The construct is engineered to express the peptide, which corresponds to the native protein sequence. The “relatively short” peptide is less than 100 amino acids in length, preferably less than 75 amino acids in length, and more preferably less than 50 amino acids in length. The protein sequence of the vaccine composition is selected because it has maximal number of epitopes contained within the sequence. As noted herein, epitope motifs may be overlapping (i.e., frame shifted relative to one another) with frame shifted overlapping epitopes, e.g. two 9-mer epitopes can be present in a 10 amino acid peptide. Such a vaccine composition is administered for therapeutic or prophylactic purposes.
  • The vaccine composition will preferably include, for example, three CTL epitopes and at least one HTL epitope from the source antigen. Junctional sequences will be analyzed to avoid sequences containing a potentially immunodominant epitope. This polyepitopic native sequence is administered either as a peptide or as a nucleic acid sequence which encodes the peptide. Alternatively, an analog can be made of this native sequence.
  • The embodiment of this example provides for the possibility that an as yet undiscovered aspect of immune system processing will apply to the native nested sequence and thereby facilitate the production of therapeutic or prophylactic immune response-inducing vaccine compositions. Additionally such an embodiment provides for the possibility of motif-bearing epitopes for an HLA makeup that is presently unknown. Furthermore, this embodiment directs the immune response to sequences that are present in native HBV antigens. Lastly, the embodiment provides an economy of scale when producing nucleic acid vaccine compositions.
  • Related to this embodiment, computer programs can be derived which identify, in a target sequence, the greatest number of epitopes per sequence length.
  • Example 14 Polyepitopic Vaccine Compositions Directed to Multiple Diseases
  • The HBV peptide epitopes of the present invention are used in conjunction with peptide epitopes from target antigens related to one or more other diseases, to create a vaccine composition that is useful for the prevention or treatment of HBV as well as another disease. Examples of other diseases include, but are not limited to, HIV, HCV, and HPV.
  • For example, a polyepitopic peptide composition comprising multiple CTL and HTL epitopes that target greater than 98% of the population may be created for administration to individuals at risk for both HBV and HIV infection. The composition can be provided as a single polypeptide that incorporates the multiple epitopes from the various disease-associated sources.
  • Example 15 Use of Peptides to Evaluate an Immune Response
  • Peptides of the invention may be used to analyze an immune response for the presence of specific CTL populations corresponding to HBV. Such an analysis may be performed as described by Ogg et al., Science 279:2103-2106, 1998. In the following example, peptides in accordance with the invention are used as a reagent for diagnostic or prognostic purposes, not as an immunogen.
  • In this example highly sensitive human leukocyte antigen tetrameric complexes (“tetramers”) may be used for a cross-sectional analysis of, for example, HBV Env-specific CTL frequencies from untreated HLA A*0201-positive individuals at different stages of infection using an HBV Env peptide containing an A2.1 extended motif. Tetrameric complexes are synthesized as described (Musey et al., N. Engl. J. Med. 337:1267, 1997). Briefly, purified HLA heavy chain (A2.1 in this example) and β2-microglobulin are synthesized by means of a prokaryotic expression system. The heavy chain is modified by deletion of the transmembrane-cytosolic tail and COOH-terminal addition of a sequence containing a BirA enzymatic biotinylation site. The heavy chain, β2-microglobulin, and peptide are refolded by dilution. The 45-kD refolded product isolated by fast protein liquid chromatography and then biotinylated by BirA in the presence of biotin (Sigma, St. Louis, Mo.), adenosine 5′ triphosphate and magnesium. Streptavidin-phycoerythrin conjugate is added in a 1:4 molar ratio, and the tetrameric product is concentrated to 1 mg/ml. The resulting product is referred to as tetramer-phycoerythrin.
  • Approximately one million PBMCs are centrifuged at 300 g for 5 minutes and resuspended in 50 ul of cold phosphate-buffered saline. Tri-color analysis is performed with the tetramer-phycoerythrin, along with anti-CD8-Tricolor, and anti-CD38. The PBMCs are incubated with tetramer and antibodies on ice for 30 to 60 min and then washed twice before formaldehyde fixation. Gates are applied to contain >99.98% of control samples. Controls for the tetramers include both A*0201-negative individuals and A*0201-positive uninfected donors. The percentage of cells stained with the tetramer is then determined by flow cytometry. The results indicate the number of cells in the PBMC sample that contain epitope-restricted CTLs, thereby readily indicating the stage of infection with HBV or the status of exposure to HBV or to a vaccine that elicits a protective response.
  • Example 16 Use of Peptide Epitopes to Evaluate Recall Responses
  • The peptide epitopes of the invention are used as reagents to evaluate T cell responses such as acute or recall responses, in patients. Such an analysis may be performed on patients who have recovered from infection or who are chronically infected with HBV or who have been vaccinated with an HBV vaccine.
  • For example, the class I restricted CTL response of persons at risk for HBV infection who have been vaccinated may be analyzed. The vaccine may be any BBV vaccine. PBMC are collected from vaccinated individuals and HLA typed. Appropriate peptide reagents that, are highly conserved and, optimally, bear supermotifs to provide cross-reactivity with multiple HLA supertype family members are then used for analysis of samples derived from individuals who bear that HLA type.
  • PBMC from vaccinated individuals are separated on Ficoll-Histopaque density gradients (Sigma Chemical Co., St. Louis, Mo.), washed three times in HBSS (GIBCO Laboratories), resuspended in RPMI-1640 (GIBCO Laboratories) supplemented with L-glutamine (2 mM), penicillin (50 U/ml), streptomycin (50 μg/m1), and Hepes (10 mM) containing 10% heat-inactivated human AB serum (complete RPMI) and plated using microculture formats. Synthetic peptide is added at 10 μg/ml to each well and recombinant HBc Ag is added at 1 μg/ml to each well as a source of T cell help during the first week of stimulation.
  • In the microculture format, 4×105 PBMC are stimulated with peptide in 8 replicate cultures in 96-well round bottom plate in 1000/well of complete RPMI. On days 3 and 10, 100 ml of complete RPMI and 20 U/ml final concentration of rIL-2 are added to each well. On day 7 the cultures are transferred into a 96-well flat-bottom plate and restimualted with peptide, rIL-2 and 105 irradiated (3,000 rad) autologous feeder cells. The cultures are tested for cytotoxic activity on day 14. A positive CTL response requires two or more of the eight replicate cultures to display greater than 10% specific 51Cr release, based on comparison with uninfected control subjects as previously described (Rehermann, et al., Nature Med. 2:1104, 1108, 1996; Rehermann et al., J. Clin. Invest. 97:1655-1665, 1996; and Rehermann et al. J. Clin. Invest. 98:1432-1440, 1996).
  • Target cell lines are autologous and allogeneic EBV-transformed B-LCL that are either purchased from the American Society for Histocompatibility and Immunogenetics (ASHI, Boston, Mass.) or established from the pool of patients as described (Guilhot, et al. J. Virol. 66:2670-2678, 1992).
  • Cytotoxicity assays are performed in the following manner. Target cells consist of either allogeneic HLA-matched or autologous EBV-transformed B lymphoblastoid cell line that are incubated overnight with synthetic peptide at 10<M and labeled with 100<Ci of 51Cr (Amersham Corp., Arlington Heights, Ill.) for 1 hour after which they are washed four times with HBSS. Cytolytic activity is determined in a standard 4-h, split well 51Cr release assay using U-bottomed 96 well plates containing 3,000 targets/well. Stimulated PBMC are tested at E/T ratios of 20-50:1 on day 14. Percent cytotoxicity is determined from the formula: 100×[(experimental release−spontaneous release)/maximum release−spontaneous release)]. Maximum release is determined by lysis of targets by detergent (2% Triton X-100®; Sigma Chemical Co., St. Louis, Mo.). Spontaneous release is <25% of maximum release for all experiments. The results of such an analysis will indicate to what extent HLA-restricted CTL populations have been stimulated with the vaccine. Of course, this protocol can also be used to monitor prior HBV exposure.
  • The above examples are provided to illustrate the invention but not to limit its scope. For example, the human terminology for the Major Histocompatibility Complex, namely HLA, is used throughout this document. It is to be appreciated that these principles can be extended to other species as well. Moreover, peptide epitopes have been disclosed in the related application U.S. Ser. No. 08/820,360, which was previously incorporated by reference. Thus, other variants of the invention will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims. All publications, patents, and patent application cited herein are hereby incorporated by reference for all purposes.
  • TABLE I
    POSITION POSTION POSITION
    2 (Primary Anchor) 3 (Primary Anchor) C Terminus (Primary Anchor)
    SUPERMOTIF
    A1 T, I, L, V, M, S F, W, Y
    A2 L, I, V, M, A, T, Q I, V, M, A, T, L
    A3 V, S, M, A, T, L, I R, K
    A24 Y, F, W, I, V, L, M, T F, I, Y, W, L, M
    B7 P V, I, L, F, M, W, Y, A
    B27 R, H, K F, Y, L, W, M, I
    B44 E, D F, W, Y, L, I, M, V, A
    B58 A, T, S F, W, Y, L, I, V
    B62 Q, L, I, V, M, P F, W, Y, M, I, V
    MOTIF
    A1 T, S, M Y
    A1 D, E, A, S Y
    A2.1 L, M, V, Q, I, A, T V, L, I, M, A, T
    A3 L, M, V, I, S, A, T, F, C, G, D K, Y, R, H, F, A
    A11 V, T, M, L, I, S, A, G, N, C, D, F K, R, Y, H
    A24 Y, F, W, M F, L, I, W
    A*3101 M, V, T, A, L, I, S R, K
    A*3301 M, V, A, L, F, I, S, T R,
    Figure US20100068228A1-20100318-P00001
    A*6801 A, V, T, M, S, L, I R,
    Figure US20100068228A1-20100318-P00001
    B*0702 P L, M, F, W Y, A, I, V
    B*3501 P L, M, F, W, Y, I, V, A
    B51 P L, I, V, F, W, Y, A, M
    B*5301 P I, M, F, W, Y, A, L, V
    B*5401 P A, T, I, V, L, M, F, W, Y
    Bold residues are preferred, italicized residues are less preferred. A peptide is considered motif-bearing if it has primary anchors at each primary anchor position for a motif or supermotif as specified in the above table.
  • TABLE II
    POSITION
    Figure US20100068228A1-20100318-P00002
    Figure US20100068228A1-20100318-P00003
    Figure US20100068228A1-20100318-P00004
    Figure US20100068228A1-20100318-P00005
    Figure US20100068228A1-20100318-P00006
    Figure US20100068228A1-20100318-P00007
    Figure US20100068228A1-20100318-P00008
    Figure US20100068228A1-20100318-P00009
    C-terminus
    SUPERMOTIFS
    A1 1° Anchor 1° Anchor
    T, I, L, V, M, S F, W, Y
    A2 1° Anchor 1° Anchor
    L, I, V, M, A, T, Q L, I, V, M, A, T
    A3 preferred 1° Anchor Y, F, W (4/5) Y, F, W (3/5) Y, F, W (4/5) P (4/5) 1°Anchor
    V, S, M, A, T, R, K
    L, I
    deleterious D, E (3/5); P (5/5) D, E (4/5)
    A24 1° Anchor 1° Anchor
    Y, F, W, I, V, L, M, T F, I, Y, W, L, M
    B7 preferred F, W, Y (5/5) 1°Anchor F, W, Y (4/5) F, W, Y 1°Anchor
    L, I, V, M (3/5) P (3/5) V, I, L, F, M, W, Y, A
    1° Anchor 1° Anchor
    deleterious D, E (3/5); P(5/5); D, E (3/5) G (4/5) Q, N (4/5) D, E
    G(4/5); A(3/5); Q, N (3/5) (4/5)
    B27 1° Anchor 1° Anchor
    R, H, K F, Y, L, W, M, I
    B44 1° Anchor 1° Anchor
    E, D F, W, Y, L, I, M, V, A
    B58 1° Anchor 1° Anchor
    A, T, S F, W, Y, L, I, V
    B62 1° Anchor 1° Anchor
    Q, L, I, V, M, P F, W, Y, M, I, V
    MOTIFS
    A1 preferred G, F, Y, W 1°Anchor D, E, A Y, F, W P D, E, Q, N Y, F, W 1°Anchor
    9-mer S, T, M Y
    deleterious D, E R, H, K, L, I, V, A G A
    M, P
    A1 preferred G, R, H, K A, S, T, C, L, I, 1°Anchor G, S, T, C A, S, T, C L, I, V, M D, E 1°Anchor
    9-mer V, M D, E, A, S Y
    deleterious A R, H, K, D, E, D, E P, Q, N R, H, K P, G G, P
    P, Y, F, W
    POSITION
    Figure US20100068228A1-20100318-P00010
    or
    Figure US20100068228A1-20100318-P00002
    Figure US20100068228A1-20100318-P00003
    Figure US20100068228A1-20100318-P00004
    Figure US20100068228A1-20100318-P00005
    Figure US20100068228A1-20100318-P00006
    Figure US20100068228A1-20100318-P00007
    Figure US20100068228A1-20100318-P00008
    Figure US20100068228A1-20100318-P00009
    C-terminus C-terminus
    A1 preferred Y, F, W 1°Anchor D, E, A, Q, N A Y, F, W, Q, N P, A, S, T, C G, D, E P 1°Anchor
    10-mer S, T, M Y
    deleterious G, P R, H, K, G, L, I, D, E R, H, K Q, N, A R, H, K, Y, F, W R, H, K A
    V, M
    A1 preferred Y, F, W S, T, C, L, I, V, M 1°Anchor A Y, F, W P, G G Y, F, W 1°Anchor
    10-mer D, E, A, S Y
    deleterious R, H, K R, H, K, D, E, P G P, R, H, K Q, N
    P, Y, F, W
    A2.1 preferred Y, F, W 1°Anchor Y, F, W S, T, C Y, F, W A P 1°Anchor
    9-mer L, M, I, V, Q, V, L, I, M, A, T
    deleterious D, E, P D, E, R, K, H R, K, H D, E, R, K, H
    A2.1 preferred A, Y, F, W 1°Anchor L, V, I, M G G F, Y, W, 1°Anchor
    10-mer L, M, I, V, Q, L, V, I, M V, L, I, M, A, T
    A, T
    deleterious D, E, P D, E R, K, H, A P R, K, H D, E, R, R, K, H
    K, H
    A3 preferred R, H, K 1°Anchor Y, F, W P, R, H, K, Y, F, W A Y, F, W P 1°Anchor
    L, M, V, I, S, K, Y, R, H, F, A
    A, T, F, C, G, D
    deleterious D, E, P D, E
    A11 preferred A 1°Anchor Y, F, W Y, F, W A Y, F, W Y, F, W P 1°Anchor
    V, T, L, M, I, K, R, Y, H
    S, A, G, N, C,
    D, F
    deleterious D, E, P A G
    A24 preferred Y, F, W, R, H, K 1°Anchor S, T, C Y, F, W Y, F, W 1°Anchor
    9-mer Y, F, W, M F, L, I, W
    deleterious D, E, G D, E G Q, N, P D, E, R, H, K G A, Q, N
    A24 preferred 1°Anchor P Y, F, W, P P 1°Anchor
    10-mer Y, F, W, M F, L, I, W
    deleterious G, D, E Q, N R, H, K D, E A Q, N D, E, A
    A3101 preferred R, H, K 1°Anchor Y, F, W P Y, F, W Y, F, W A, P 1°Anchor
    M, V, T, A, L, R, K
    I, S
    deleterious D, E, P D, E A, D, E D, E D, E D, E
    A3301 preferred 1°Anchor Y, F, W A, Y, F, W 1°Anchor
    M, V, A, L, F, R, K
    I, S, T
    deleterious G, P D, E
    A6801 preferred Y, F, W, S, T, C 1°Anchor Y, F, W, L, I, Y, F, W P 1°Anchor
    A, V, T, M, S, V, M R, K
    L, I
    deleterious G, P D, E, G R, H, K A
    B0702 preferred R, H, K, F, W, Y 1°Anchor R, H, K R, H, K R, H, K R, H, K P, A 1°Anchor
    P L, M, F, W, Y, A,
    I, V
    deleterious D, E, Q, N, P D, E, P D, E D, E G, D, E Q, N D, E
    B3501 preferred F, W, Y, L, I, V, 1°Anchor F, W, Y F, W, Y 1°Anchor
    M P L, M, F, W, Y, I,
    V, A
    deleterious A, G, P G G
    B51 preferred L, I, V, M, F, W, 1°Anchor F, W, Y S, T, C F, W, Y G F, W, Y 1°Anchor
    Y P L, I, V, F, W, Y,
    A, M
    deleterious A, G, P, D, E, R, D, E G D, E, Q, N G, D, E
    H, K, S, T, C
    B5301 preferred L, I, V, M, F, W, 1°Anchor F, W, Y S, T, C F, W, Y L, I, V, M, F, F, W, Y 1°Anchor
    Y P W, Y I, M, F, W, Y, A,
    L, V
    deleterious A, G, P, Q, N G R, H, K, Q, N D, E
    B5401 preferred F, W, Y 1°Anchor F, W, Y, L, I, L, I, V, M A, L, I, V, M F, W, Y, A, P 1°Anchor
    P V, M A, T, I, V, L, M,
    F, W, Y
    deleterious G, P, Q, N, D, E G, D, E, S, T, C R, H, K, D, E D, E Q, N, D, G, E D, E
    Italicized residues indicate less preferred or “tolerated” residues.
    The information in Table II is specific for 9-mers unless otherwise specified.
  • TABLE III
    SEQ ID POSITION
    NO: MOTIFS
    Figure US20100068228A1-20100318-P00011
    Figure US20100068228A1-20100318-P00003
    Figure US20100068228A1-20100318-P00004
    Figure US20100068228A1-20100318-P00005
    Figure US20100068228A1-20100318-P00006
    Figure US20100068228A1-20100318-P00012
    Figure US20100068228A1-20100318-P00008
    Figure US20100068228A1-20100318-P00009
    Figure US20100068228A1-20100318-P00010
    DR4 preferred F, M, Y, L, I, M T I V, S, T, C, P, A, M, H M, H
    V, W L, I, M
    deleterious W R W, D, E
    DR1 preferred M, F, L, I, V, P, A, M, Q V, M, A, T, S, P, M A, V, M
    W, Y L, I, C,
    deleterious C C, H F, D C, W, D G, D, E D
    2578 DR7 preferred M, F, L, I, V, M W A, I, V, M, S, A, C, M I, V
    W, Y T, P, L,
    2579 deleterious C G, G, R, D N G
    DR Supermotif M, F, L, I, V, V, M, S, T, A, C,
    W, Y P, L, I
    DR3 MOTIFS
    Figure US20100068228A1-20100318-P00011
    Figure US20100068228A1-20100318-P00003
    Figure US20100068228A1-20100318-P00004
    Figure US20100068228A1-20100318-P00013
    Figure US20100068228A1-20100318-P00006
    Figure US20100068228A1-20100318-P00012
    motif a L, I, V, M, F, Y D
    preferred
    motif b L, I, V, M, F, D, N, Q, E, K, R, H
    preferred A, Y S, T
    Italicized residues indicate less preferred or “tolerated” residues.
  • TABLE IV
    HLA Class I Standard Peptide Binding Affinity.
    STANDARD SEQ
    STANDARD BINDING ID
    ALLELE PEPTIDE SEQUENCE AFFINITY (nM) NO:
    A*0101 944.02 YLEPAIAKY 25 2486
    A*0201 941.01 FLPSDYFPSV 5.0 2487
    A*0202 941.01 FLPSDYFPSV 4.3 2487
    A*0203 941.01 FLPSDYFPSV 10 2487
    A*0206 941.01 FLPSDYFPSV 3.7 2487
    A*0207 941.01 FLPSDYFPSV 23 2487
    A*6802 1072.34 YVIKVSARV 40 2488
    A*0301 941.12 KVFPYALINK 11 2489
    A*1101 940.06 AVDLYHFLK 6.0 2490
    A*3101 941.12 KVFPYALINK 18 2489
    A*3301 1083.02 STLPETYVVRR 29 2491
    A*6801 941.12 KVFPYALINK 8.0 2489
    A*2401 979.02 AYIDNYNKF 12 2492
    B*0702 1075.23 APRTLVYLL 5.5 2493
    B*3501 1021.05 FPFKYAAAF 7.2 2494
    B51 1021.05 FPFKYAAAF 5.5 2494
    B*5301 1021.05 FPFKYAAAF 9.3 2494
    B*5401 1021.05 FPFKYAAAF 10 2494
  • TABLE V
    HLA Class II Standard Peptide Binding Affinity.
    Binding
    Standard Affinity SEQ ID
    Allele Nomenclature Peptide Sequence (nM) NO:
    DRB1*0101 DR1 515.01 PKYVKQNTLKLAT 5.0 2495
    DRB1*0301 DR3 829.02 YKTIAFDEEARR 300 2496
    DRB1*0401 DR4w4 515.01 PKYVKQNTLKLAT 45 2495
    DRB1*0404 DR4w14 717.01 YARFQSQTTLKQKT 50 2497
    DRB1*0405 DR4w15 717.01 YARFQSQTTLKQKT 38 2497
    DRB1*0701 DR7 553.01 QYIKANSKFIGITE 25 2498
    DRB1*0802 DR8w2 553.01 QYIKANSKFIGITE 49 2498
    DRB1*0803 DR8w3 553.01 QYIKANSKFIGITE 1600 2498
    DRB1*0901 DR9 553.01 QYIKANSKFIGITE 75 2498
    DRB1*1101 DRSw11 553.01 QYIKANSKFIGITE 20 2498
    DRB1*1201 DR5w12 1200.05 EALIHQLKINPYVLS 298 2499
    DRB1*1302 DR6w19 650.22 QYIKANAKFIGITE 3.5 2500
    DRB1*1501 DR2w2β1 507.02 GRTQDENPVVHFFK 9.1 2501
    NIVTPRTPPP
    DRB3*0101 DR52a 511 NGQIGNDPNRDIL 470 2502
    DRB4*0101 DRw53 717.01 YARFQSQTTLKQKT 58 2503
    DRB5*0101 DR2wβ2 553.01 QYIKANSKFIGITE 20 2504
    The “Nomenclature” column lists the allelic designations used in Table XVIII.
  • TABLE VI
    HBV A01 SUPER MOTIF(With binding information)
    SEQ ID
    Conservancy Freq. Protein Position Sequence String Peptide Filed A*0101 NO:
    95 19 POL 521 AICSVVRRAF XIXXXXXXXF 1
    95 19 NUC 54 ALRQAILCW XLXXXXXXW 2
    80 16 ENV 108 AMQWNSTTF XMXXXXXXF 3
    100 20 POL 166 ASFCGSPY XSXXXXXY 26.0026 * 4
    100 20 POL 166 ASFCGSPYSW XSXXXXXXXW 5
    90 18 NUC 19 ASKLCLGW XSXXXXXW 6
    85 17 NUC 19 ASKLCLGWLW XSXXXXXXXW 7
    80 16 POL 822 ASPLHVAW XSXXXXXW 8
    100 20 ENV 312 CIPIPSSW XIXXXXXW 9
    100 20 ENV 312 CIPIPSSWAF XIXXXXXXXF 10
    95 19 ENV 253 CLIFLLVLLDY XLXXXXXXXXY 26.0548 11
    95 19 ENV 239 CLRRFIIF XLXXXXXF 12
    75 15 ENV 239 CLRRFIIFLF XLXXXXXXXF 13
    95 19 POL 523 CSVVRRAF XSXXXXXF 14
    100 20 ENV 310 CTCIPIPSSW XTXXXXXXXW 15
    90 18 NUC 31 DIDPYKEF XIXXXXXF 16
    85 17 NUC 29 DLLDTASALY XLXXXXXXXY 1.0519 * 11.1000 17
    95 19 ENV 196 DSWWTSLNF XSXXXXXXF 20.0120 18
    95 19 NUC 43 ELLSFLPSDF XLXXXXXXXF 19
    95 19 NUC 43 ELLSFLPSDFF XLXXXXXXXXF 20
    95 19 POL 374 ESRLVVDF XSXXXXXF 21
    95 19 POL 374 ESRLVVDFSQF XSXXXXXXXXF 22
    80 16 ENV 248 FILLLCLIF XIXXXXXXF 23
    80 16 ENV 246 FLFILLLCLIF XLXXXXXXXXF 24
    95 19 ENV 256 FLLVLLDY XLXXXXXY 26.0027 25
    95 19 POL 658 FSPTYKAF XSXXXXXF 26
    90 18 X 63 FSSAGPCALRF XSXXXXXXXXF 27
    100 20 ENV 333 FSWLSLLVPF XSXXXXXXXF 20.0263 28
    95 19 POL 656 FTFSPTYKAF XTXXXXXXXF 20.0262 29
    95 19 ENV 346 FVGLSPTVW XVXXXXXXW 30
    95 19 POL 627 GLLGFAAPF XLXXXXXXF 20.0124 31
    95 19 POL 509 GLSPFLLAQF XLXXXXXXXF 32
    85 17 NUC 29 GMDIDPYKEF XMXXXXXXXF 26.0372 33
    95 19 NUC 123 GVWIRTPPAY XVXXXXXXXY 1.0525 0.0017 34
    75 15 POL 569 HLNPNKTKRW XLXXXXXXXW 35
    80 16 POL 491 HLYSHPIILGF XLXXXXXXXXF 36
    85 17 POL 715 HTAELLAACF XTXXXXXXXF 37
    95 19 NUC 52 HTALRQAILCW XTXXXXXXXXW 38
    100 20 POL 149 HTLWKAGILY XTXXXXXXXY 1.0542 * 0.0300 39
    100 20 ENV 249 ILLLCLIF XLXXXXXF 40
    80 16 POL 760 ILRGTSFVY XLXXXXXXY 1.0205 * 0.0017 41
    90 18 ENV 188 ILTIPQSLDSW XLXXXXXXXXW 42
    90 18 POL 625 IVGLLGFAAPF XVXXXXXXXXF 43
    80 16 POL 503 KIPMGVGLSPF XIXXXXXXXXF 44
    85 17 NUC 21 KLCLGWLW XLXXXXXW 45
    75 15 POL 108 KLIMPARF XLXXXXXF 45
    75 15 POL 108 KLIMPARFY XLXXXXXXY 1.0171 0.0017 47
    80 16 POL 610 KLPVNRPIDW XLXXXXXXXW 48
    85 17 POL 574 KTKRWGYSLNF XTXXXXXXXXF 49
    95 19 POL 55 KVGNFTGLY XVXXXXXXY 1.0166 * 0.0680 50
    95 19 ENV 254 LIFLLVLLDY XIXXXXXXXY 1.0899 0.0084 51
    100 20 POL 109 LIMPARFY XIXXXXXY 26.0028 52
    85 17 NUC 30 LLDTASALY XLXXXXXXY 1.0155 * 25.0000 53
    80 16 POL 752 LLGCAANW XLXXXXXW 54
    95 19 POL 628 LLGFAAPF XLXXXXXF 55
    100 20 ENV 378 LLPIFFCLW XLXXXXXXW 56
    100 20 ENV 378 LLPIFFCLWVY XLXXXXXXXXY 26.0549 * 57
    95 19 NUC 44 LLSFLPSDF XLXXXXXXF 58
    95 19 NUC 44 LLSFLPSDFF XLXXXXXXXF 59
    90 18 POL 407 LLSSNLSW XLXXXXXW 60
    95 19 ENV 175 LLVLQAGF XLXXXXXF 61
    95 19 ENV 175 LLVLQAGFF XLXXXXXXF 20.0121 62
    100 20 ENV 338 LLVPFVQW XLXXXXXW 63
    100 20 ENV 338 LLVPFVQWF XLXXXXXXF 64
    85 17 NUC 100 LLWFHISCLTF XLXXXXXXXXF 65
    95 19 NUC 45 LSFLPSDF XSXXXXXF 66
    95 19 NUC 45 LSFLPSDFF XSXXXXXXF 20.0123 67
    95 19 POL 415 LSLDVSAAF XSXXXXXXF 68
    95 19 POL 415 LSLDVSAAFY XSXXXXXXXY 2.0239 * 4.2000 69
    100 20 ENV 336 LSLLVPFVQW XSXXXXXXXW 70
    100 20 ENV 336 LSLLVPFVQWF XSXXXXXXXXF 71
    95 19 X 53 LSLRGLPVCAF XSXXXXXXXXF 72
    95 19 POL 510 LSPFLLAQF XSXXXXXXF 73
    75 15 ENV 349 LSPTVWLSVIW XSXXXXXXXXW 74
    85 17 POL 742 LSRKYTSF XSXXXXXF 75
    85 17 POL 742 LSRKYTSFPW XSXXXXXXXW 76
    75 15 ENV 16 LSVPNPLGF XSXXXXXXF 77
    75 15 NUC 137 LTFGRETVLEY XTXXXXXXXXY 78
    90 18 ENV 189 LTIPQSLDSW XTXXXXXXXW 79
    90 18 ENV 189 LTIPQSLDSWW XTXXXXXXXXW 80
    90 18 POL 404 LTNLLSSNLSW XTXXXXXXXXW 81
    95 19 ENV 176 LVLQAGFF XVXXXXXF 82
    100 20 ENV 339 LVPFVQWF XVXXXXXF 83
    100 20 POL 377 LVVDFSQF XVXXXXXF 84
    85 17 ENV 360 MMWYWGPSLY XMXXXXXXXY 1039.01 * 0.0810 85
    75 15 X 103 MSTTDLEAY XSXXXXXXY 2.0126 * 0.8500 86
    75 15 X 103 MSTTDLEAYF XSXXXXXXXF 87
    95 19 POL 42 NLGNLNVSIPW XLXXXXXXXXW 88
    90 18 POL 406 NLLSSNLSW XLXXXXXXW 89
    95 19 POL 45 NLNVSIPW XLXXXXXW 90
    75 15 ENV 15 NLSVPNPLGF XLXXXXXXXF 91
    90 18 POL 738 NSVVLSRKY XSXXXXXXY 2.0123 0.0005 92
    100 20 ENV 380 PIFFCLWVY XIXXXXXXY 1.0843 0.0078 93
    100 20 ENV 314 PIPSSWAF XIXXXXXF 94
    100 20 POL 124 PLDKGIKPY XLXXXXXXY 1.0174 * 0.0190 95
    100 20 POL 124 PLDKGIKPYY XLXXXXXXXY 1.0541 * 0.1600 96
    100 20 ENV 377 PLLPIFFCLW XLXXXXXXXW 97
    95 19 ENV 174 PLLVLQAGF XLXXXXXXF 98
    95 19 ENV 174 PLLVLQAGFF XLXXXXXXXF 99
    80 16 POL 505 PMGVGLSPF XMXXXXXXF 100
    85 17 POL 797 PTTGRTSLY XTXXXXXXY 1.0208 * 0.7700 101
    75 15 ENV 351 PTVWLSVIW XTXXXXXXW 102
    85 17 POL 612 PVNRPIDW XVXXXXXW 103
    95 19 POL 685 QVFADATPTG XVXXXXXXXXW 104
    90 18 POL 624 RIVGLLGF XIXXXXXF 105
    75 15 POL 106 RLKLIMPARF XLXXXXXXXF 106
    75 15 POL 106 RLKLIMPARFY XLXXXXXXXXY 107
    95 19 POL 376 RLVVDFSQF XLXXXXXXF 20.0122 108
    90 18 POL 353 RTPARVTGGVF XTXXXXXXXXF 109
    100 20 POL 49 SIPWTHKVGNF XIXXXXXXXXF 110
    95 19 ENV 194 SLDSWWTSLNF XLXXXXXXXXF 111
    95 19 POL 416 SLDVSAAF XLXXXXXF 112
    95 19 POL 416 SLDVSAAFY XLXXXXXXY 1.0186 * 17.2000 113
    100 20 ENV 337 SLLVPFVQW XLXXXXXXW 114
    100 20 ENV 337 SLLVPFVQWF XLXXXXXXXF 115
    95 19 X 54 SLRGLPVCAF XLXXXXXXXF 20.0259 116
    90 18 X 64 SSAGPCALRF XSXXXXXXXF 26.0374 117
    75 15 X 104 STTDLEAY XTXXXXXY 118
    75 15 X 104 STTDLEAYF XTXXXXXXF 119
    75 15 ENV 17 SVPNPLGF XVXXXXXF 120
    90 18 POL 739 SVVLSRKY XVXXXXXY 26.0029 121
    85 17 POL 739 SVVLSRKYTSF XVXXXXXXXXF 122
    90 18 ENV 190 TIPQSLDSW XIXXXXXXW 123
    90 18 ENV 190 TIPQSLDSWW XIXXXXXXXW 124
    100 20 POL 150 TLWKAGILY XLXXXXXXY 1.0177 * 0.0017 125
    75 15 X 105 TTDLEAYF XTXXXXXF 126
    85 17 POL 798 TTGRTSLY XTXXXXXY 26.0030 127
    80 16 NUC 16 TVQASKLCLGW XVXXXXXXXXW 128
    75 15 ENV 352 TVWLSVIW XVXXXXXW 129
    85 17 POL 741 VLSRKYTSF XLXXXXXXF 130
    85 17 POL 741 VLSRKYTSFPW XLXXXXXXXXW 131
    85 17 POL 740 VVLSRKYTSF XVXXXXXXXF 20.0261 132
    80 16 POL 759 WILRGTSF XIXXXXXF 133
    80 16 POL 759 WILRGTSFVY XIXXXXXXXY 1.0572 0.0023 134
    95 19 NUC 125 WIRTPPAY XIXXXXXY 26.0031 135
    80 16 POL 751 WLLGCAANW XLXXXXXXW 136
    95 19 POL 414 WLSLDVSAAF XLXXXXXXXF 137
    95 19 POL 414 WLSLDVSAAFY XLXXXXXXXXY 26.0551 138
    100 20 ENV 335 WLSLLVPF XLXXXXXF 139
    100 20 ENV 335 WLSLLVPFVQW XLXXXXXXXXW 140
    85 17 NUC 26 WLWGMDIDPY XLXXXXXXXY 1.0774 * 0.0810 141
    95 19 ENV 237 WMCLRRFIIF XMXXXXXXXF 20.0266 142
    85 17 ENV 359 WMMWYWGPS XMXXXXXXXXY 26.0552 * 143
    100 20 POL 52 WTHKVGNF XTXXXXXF 144
    100 20 POL 122 YLPLDKGIKPY XLXXXXXXXXY 26.0553 145
    90 18 NUC 118 YLVSFGVW XLXXXXXW 146
    80 16 POL 493 YSHPIILGF XSXXXXXXF 147
    85 17 POL 580 YSLNFMGY XSXXXXXY 26.0032 148
    148
  • TABLE VII
    HBV A02 SUPER MOTIF (With binding information)
    EQ
    Conser- Fre- Posi- C- ID
    vancy quency Protein tion Sequence P2 term Peptide AA Filed A*0201 A*0202 A*0203 A*0206 A*6802 NO:
    85 17 POL 721 AACFARSRSGA A A 11 149
    85 17 POL 431 AAMPHLLV A V 8 150
    80 16 POL 756 AANWILRGT A T 9 151
    95 19 POL 632 AAPFTQCGYPA A A 11 152
    95 19 POL 521 AICSVVRRA I A 5.0025 9 0.0001 153
    90 18 NUC 58 AILCWGEL I L 8 154
    90 18 NUC 58 AILCWGELM I M 9 155
    95 19 POL 642 ALMPLYACI L I 927.15 9 * 0.5000 0.0340 3.3000 0.2500 0.0470 156
    80 16 ENV 108 AMQWNSTT M T 8 157
    75 15 X 102 AMSTTDLEA M A 3.0051 9 0.0013 158
    95 19 POL 690 ATPTGWGL T L 8 159
    80 16 POL 690 ATPTGWGLA T A 9 160
    75 15 POL 690 ATPTGWGLAI T I 10 161
    95 19 POL 397 AVPNLQSL V L 8 162
    95 19 POL 397 AVPNLQSLT V T 5.0026 9 0.0001 163
    95 19 POL 397 AVPNLQSLTNL V L 11 164
    80 16 POL 755 CAANWILRGT A T 10 165
    95 19 X 61 CAFSSAGPCA A A 5.0090 10 0.0001 166
    95 19 X 61 CAFSSAGPCAL A L 11 167
    90 18 X 69 CALRFTSA A A 8 168
    100 20 ENV 312 CIPIPSSWA I A 5.0007 9 0.0010 169
    80 16 ENV 312 CIPIPSSWAFA I A 11 170
    90 18 POL 533 CLAFSYMDDV L V 1.0559 10 0.0008 171
    90 18 POL 533 CLAFSYMDDVV L V 11 172
    85 17 NUC 23 CLGWLWGM L M 8 173
    85 17 NUC 23 CLGWLWGMDI L I 3.0210 10 0.0093 174
    100 20 ENV 253 CLIFLLVL L L Chisari 6 0.0002 175
    4.011
    100 20 ENV 253 CLIFLLVLL L L 1.0836 9 0.0006 176
    95 19 ENV 239 CLRRFIIFL L L 1.0829 0.0002 177
    75 15 ENV 239 CLRRFIIFLFI L I Chisari 11 0.0004 178
    4.055
    90 18 NUC 107 CLTFGRET L T 8 179
    90 18 NUC 107 CLTFGRETV L V 1.0160 9 0.0001 180
    100 20 ENV 310 CTCIPIPSSWA T A 11 181
    95 19 POL 689 DATPTGWGL A L 5.0027 9 0.0001 182
    80 16 POL 689 DATPTGWGLA A A 10 183
    75 15 POL 689 DATPTGWGLAI A I 11 184
    90 18 NUC 31 DIDPYKEFGA I A 10 185
    85 17 NUC 29 DLLDTASA L A 8 186
    85 17 NUC 29 DLLDTASAL L L 1.0154 9 0.0001 187
    95 19 POL 40 DLNLGNLNV L V 927.30 9 0.0004 188
    95 19 POL 40 DLNLGNLNVSI L I 11 189
    80 16 NUC 32 DTASALYREA T A 10 190
    80 16 NUC 32 DTASALYREAL T L 11 191
    95 19 X 14 DVLCLRPV V V 8 192
    95 19 X 14 DVLCLRPVGA V A 5.0091 10 0.0001 193
    90 18 POL 541 DVVLGAKSV V V 1.0190 9 0.0003 194
    100 20 POL 17 EAGPLEEEL A L 5.0028 9 0.0001 195
    80 16 X 122 ELGEERL L L 8 196
    90 18 POL 718 ELLAACFA L A 8 197
    75 15 NUC 142 ETVLEYLV T V 8 198
    95 19 POL 687 FADATPTGWGL A L 11 199
    85 17 POL 724 FARSRSGA A A 8 200
    80 16 POL 821 FASPLHVA A A 8 201
    95 19 POL 396 FAVPNLQSL A L 9 202
    95 19 POL 396 FAVPNLQSLT A T 5.0083 10 0.0003 203
    80 16 ENV 243 FIIFLFIL I L Chisari 8 0.0006 204
    4.047
    80 16 ENV 243 FIIFLFILL I L 1.0830 9 0.0002 205
    80 16 ENV 243 FIIFLFILLL I L 1.0894 10 0.0012 206
    80 16 ENV 248 FILLLCLI I I Chisari 8 0.0003 207
    4.048
    80 16 ENV 248 FILLLCLIFL I L 1.0895 10 * 0.0280 208
    80 16 ENV 248 FILLLCLIFLL I L Chisari 11 0.0010 209
    4.049
    80 16 ENV 246 FLFILLLCL L L 1.0832 9 0.0002 210
    80 16 ENV 246 FLFILLLCLI L I 3.0206 10 0.0013 211
    75 15 ENV 171 FLGPLLVL L L 8 212
    75 15 ENV 171 FLGPLLVLQA L A 3.0205 10 * 0.0190 213
    95 19 POL 513 FLLAQFTSA L A 1069.07 9 * 0.2400 214
    95 19 POL 513 FLLAQFTSAI L I 11147.13 10 * 0.2100 0.0320 7.0000 0.1100 0.660 215
    95 19 POL 562 FLLSLGIHL L L 927.11 9 * 0.6500 0.0010 0.0100 0.1100 0.0035 216
    80 16 ENV 183 FLLTRILT L T 8 217
    80 16 ENV 183 FLLTRILTI L I 777.03 9 * 0.5100 0.0430 8.0000 0.2000 0.0010 218
    95 19 ENV 256 FLLVLLDYQGM L M 11 219
    100 20 POL 363 FLVDKNPHNT L T 5.0084 10 0.0012 220
    95 19 POL 656 FTFSPTYKA T A 1147.15 9 * 0.0056 0.0150 0.0031 0.8000 7.3000 221
    95 19 POL 656 FTFSPTYKAFL T L 11 222
    95 19 POL 59 FTGLYSST T T 6 223
    90 18 POL 59 FTGLYSSTV T V 20.0118 9 0.0005 224
    95 19 POL 635 FTQCGYPA T A 8 225
    95 19 POL 635 FTQCGYPAL T L 5.0031 9 0.0009 226
    95 19 POL 635 FTQCGYPALM T M 5.0085 10 0.0024 227
    95 19 POL 518 FTSAICSV T V 8 228
    95 19 POL 518 FTSAICSVV T V 5.0032 9 0.0090 229
    95 19 ENV 346 FVGLSPTV V V 8 230
    95 19 ENV 346 FVGLSPTVWL V L 1.0931 10 0.0008 231
    90 18 X 132 FVLGGCRHKL V L Chisari 10 0.0030 232
    4.114
    90 18 X 132 FVLGGCRHKLV V V 11 233
    95 19 ENV 342 FVQWFVGL V L 8 234
    95 19 ENV 342 FVQWFVGLSPT V T 11 235
    90 18 POL 766 FVYVPSAL V L 8 236
    90 18 POL 766 FVYVPSALNPA V A 11 237
    95 19 X 50 GAHLSLRGL A L 5.0040 9 0.0001 238
    90 18 X 50 GAHLSLRGLPV A V 11 239
    85 17 POL 545 GAKSVQHL A L 8 240
    85 17 POL 545 GAKSVQHLESL A L 11 241
    75 15 POL 567 GIHLNPNKT I T 9 242
    90 18 POL 155 GILYKRET I T 8 243
    90 18 POL 155 GILYKRETT I T 9 244
    85 17 POL 682 GLCQVFADA L A 1142.04 9 * 0.0024 245
    85 17 POL 682 GLCQVFADAT L T 10 246
    95 19 POL 627 GLLGFAAPFT L T 5.0086 10 0.0049 247
    85 17 ENV 62 GLLGWSPQA L A 1142.07 9 * 0.4000 0.0003 0.0350 0.2600 0.0005 248
    95 19 X 57 GLPVCAFSSA L A 5.0092 10 0.0008 249
    95 19 POL 509 GLSPFLLA L A 8 250
    95 19 POL 509 GLSPFLLAQFT L T 11 251
    100 20 ENV 348 GLSPTVWL L L Chisari 8 0.0036 252
    4.012
    75 15 ENV 348 GLSPTVWLSV L V 1.0518 10 * 0.2600 253
    75 15 ENV 348 GLSPTVWLSVI L I Chisari 11 0.0036 254
    4.031
    90 18 ENV 265 GMLPVCPL M L 8 255
    90 18 POL 735 GTDNSVVL T L 8 256
    75 15 ENV 13 GTNLSVPNPL T L 10 257
    80 16 POL 763 GTSFVYVPSA T A 10 258
    80 16 POL 763 GTSFVYVPSAL T L 11 259
    80 16 POL 507 GVGLSPFL V L 8 260
    80 16 POL 507 GVGLSPFLL V L Chisari 9 0.0002 261
    4.082
    80 16 POL 507 GVGLSPFLLA V A 10 262
    95 19 NUC 123 GVWIRTPPA V A 3.0040 9 0.0030 263
    90 18 NUC 104 HISCLTFGRET I T 11 264
    80 16 POL 435 HLLVGSSGL L L 927.43 9 0.0031 265
    90 18 X 52 HLSLGLPV L V 927.02 9 0.0014 266
    90 18 X 52 HLSLRGLPVCA L A 11 267
    80 16 POL 491 HLYSHPII L I 17.0256 8 268
    80 16 POL 491 HLYSHPIIL L L 927.47 9 * 0.2200 0.0003 0.9300 0.1700 0.0530 269
    85 17 POL 715 HTAELLAA T A 8 270
    85 17 POL 715 HTAELLAACFA T A 11 271
    100 20 NUC 52 HTALRQAI T I 8 272
    95 19 NUC 52 HTALRQAIL T L 5.0021 9 0.0001 273
    100 20 POL 149 HTLWKAGI T I 8 274
    100 20 POL 149 HTLWKAGIL T L 5.0033 9 0.0001 275
    80 16 ENV 244 IIFLFILL I L Chisari 8 0.0004 276
    4.051
    80 16 ENV 244 IIFLFILLL I L 1.0831 9 0.0002 277
    80 16 ENV 244 IIFLFILLLCL I L Chisari 11 0.0002 278
    4.052
    80 16 POL 497 IILGFRKI I I 8 279
    80 16 POL 497 IILGFRKIPM I M 10 280
    90 18 NUC 59 ILCWGELM L M 8 281
    80 16 POL 498 ILGFRKIPM L M 3.0016 9 0.0002 282
    100 20 ENV 249 ILLLCLIFL L L 1137.04 9 * 0.0015 283
    100 20 ENV 249 ILLLCLIFLL L L 1069.08 10 * 0.0190 0.0001 0.0002 0.1300 0.0015 284
    100 20 ENV 249 ILLLCLIFLLV L V Chisari 11 0.0056 285
    4.013
    80 16 POL 760 ILRGTSFV L V 8 286
    80 16 POL 760 ILRGTSFVYV L V 1.0573 10 0.0160 287
    100 20 NUC 139 ILSTLPET L T 8 288
    100 20 NUC 139 ILSTLPETT L T 5.0022 9 0.0001 289
    100 20 NUC 139 ILSTLPETTV L V 1069.14 10 * 0.0210 0.0085 0.0770 0.3100 0.0067 290
    100 20 NUC 139 ILSTLPETTVV L V 11 291
    95 19 ENV 188 ILTIPQSL L L 8 292
    90 18 POL 156 ILYKRETT L T 8 293
    90 18 POL 625 IVGLLGFA V A 8 294
    90 18 POL 625 IVGLLGFAA V A 3.0041 9 0.0009 295
    90 18 POL 153 KAGILYKRET A T 10 296
    90 18 POL 153 KAGILYKRETT A T 11 297
    80 16 POL 503 KIPMGVGL I L 8 298
    85 17 NUC 21 KLCLGWLWGM L M 1142.02 10 * 0.0001 299
    95 19 POL 489 KLHLYSHPI L I 927.46 9 * 0.0690 0.0340 2.7000 0.5900 0.0015 300
    80 16 POL 489 KLHLYSHPII L I 10 301
    80 16 POL 489 KLHLYSHPIIL L L 11 302
    80 16 POL 610 KLPVNRPI L I 8 303
    95 19 POL 574 KTKRWGYSL T L 5.0034 9 0.0001 304
    85 17 POL 620 KVCQRIVGL V L 1.0198 9 0.0003 305
    85 17 POL 620 KVCQRIVGLL V L 1.0567 10 0.0001 306
    95 19 POL 55 KVGNFTGL V L 17.0116 8 307
    85 17 X 91 KVLHKRTL V L 8 308
    85 17 X 91 KVLHKRTLGL V L Chisari 10 0.0004 309
    4.115
    90 18 POL 534 LAFSYMDDV A V 20.0119 9 0.0002 310
    90 18 POL 534 LAFSYMDDVV A V 20.0257 10 0.0003 311
    90 18 POL 534 LAFSYMDDVVL A L 11 312
    95 19 POL 515 LAQFTSAI A I 8 313
    95 19 POL 515 LAQFTSAICSV A V 11 314
    100 20 ENV 254 LIFLLVLL I L Chisari 8 0.0025 315
    4.014
    95 19 POL 514 LLAQFTSA L A 8 316
    95 19 POL 514 LLAQFTSAI L I 1069.05 9 * 0.1000 0.2700 3.7000 0.2600 0.7900 317
    100 20 ENV 251 LLCLIFLL L L Chisari 8 0.0004 318
    4.015
    100 20 ENV 251 LLCLIFLLV L V 1137.03 9 * 0.0048 319
    100 20 ENV 251 LLCLIFLLVL L L 1.0898 10 0.0075 320
    100 20 ENV 251 LLCLIFLLVLL L L Chisari 11 0.0013 321
    4.016
    85 17 NUC 30 LLDTASAL L L 8 . 322
    95 19 ENV 260 LLDYQGML L L Chisari 8 0.0004 323
    4.021
    90 18 ENV 260 LLDYQGMLPV L V 1137.02 10 * 0.0980 0.0001 0.0200 0.6700 0.0009 324
    80 16 POL 752 LLGCAANWI L I 927.22 9 0.0011 325
    80 16 POL 752 LLGCAANWIL L L 1.0912 10 * 0.0140 326
    95 19 POL 628 LLGFAAPFT L T 5.0035 9 0.0008 327
    85 17 ENV 63 LLGWSPQA L A 8 328
    75 15 ENV 63 LLGWSPQAQGI L I 11 329
    100 20 ENV 250 LLLCLIFL L L Chisari 8 0.0006 330
    4.017
    100 20 ENV 250 LLLCLIFLL L L 1090.05 9 * 0.0085 331
    100 20 ENV 250 LLLCLIFLLV L V 1137.01 10 * 0.0036 332
    100 20 ENV 250 LLLCLIFLLVL L L Chisari 11 0.0005 333
    4.018
    100 20 ENV 378 LLPIFFCL L L Chisari 8 0.0055 334
    4.019
    100 20 ENV 378 LLPIFFCLWV L V 1069.10 10 * 0.0320 0.0008 0.0150 0.8000 0.0005 335
    95 19 POL 563 LLSLGIHL L L 8 336
    90 18 POL 407 LLSSNLSWL L L 927.41 9 * 0.0110 0.0780 3.9000 0.2700 0.0100 337
    90 18 POL 407 LLSSNLSWLSL L L 11 338
    80 16 ENV 184 LLTRILTI L I Chisari 8 0.0026 339
    4.053
    80 16 POL 436 LLVGSSGL L L 8 340
    95 19 ENV 257 LLVLLDYQGM L M 3.0207 10 0.0050 341
    95 19 ENV 257 LLVLLDYQGML L L 11 342
    90 18 ENV 175 LLVLOAGFFL L L 1090.06 10 0.0310 0.0037 0.0045 0.1500 0.0110 343
    90 18 ENV 175 LLVLQAGFFLL L L Chisari 11 0.0074 344
    4.028
    95 19 ENV 338 LLVPFVQWFV L V 1069.06 10 * 0.6700 0.3800 1.7000 0.2900 0.1400 345
    90 18 NUC 100 LLWFHISCL L L 1142.01 9 * 0.0130 0.0002 0.0420 0.3100 0.0098 346
    85 17 NUC 100 LLWFHISCLT L T 10 347
    95 19 POL 643 LMPLYACI M I 17.0130 8 348
    95 19 NUC 108 LTFGRETV T V 8 349
    75 15 NUC 137 LTFGRETVL T L 9 350
    90 18 POL 404 LTNLLSSNL T L 9 351
    80 16 ENV 185 LTRILTIPQSL T L 11 352
    85 17 POL 99 LTVNEKRRL T L 9 353
    100 20 POL 364 LVDKNPHNT V T 5.0036 9 0.0001 354
    95 19 ENV 258 LVLLDYQGM V M 3.0034 9 0.0001 355
    95 19 ENV 258 LVLLDYQGML V L 1.0515 10 0.0001 356
    90 18 ENV 176 LVLQAGFFL V L 1.0827 9 0.0096 357
    90 18 ENV 176 LVLQAGFFLL V L 1132.17 10 * 0.0022 358
    90 18 ENV 176 LVLQAGFFLLT V T 11 359
    95 19 ENV 339 LVPFVQWFV V V 1132.01 9 * 0.0420 0.0150 0.0048 0.7900 2.8000 360
    95 19 ENV 339 LVPFVQWFVGL V L 11 361
    90 18 NUC 119 LVSFGVWI V I Chisari 8 0.0004 362
    4.078
    90 18 NUC 119 LVSFGVWIRT V T 10 363
    85 17 ENV 360 MMWYWGPSL M L 1039.03 9 * 0.6400 364
    100 20 NUC 136 NAPILSTL A L 8 365
    100 20 NUC 136 NAPILSTLPET A T 11 366
    95 19 POL 42 NLGNLNVSI L I 3.0008 9 0.0047 367
    90 18 POL 406 NLLSSNLSWL L L 1.0549 10 0.0016 368
    95 19 POL 45 NLNVSIPWT L T 5.0037 9 0.0005 369
    100 20 POL 400 NLQSLTNL L L 8 370
    100 20 POL 400 NLQSLTNLL L L 927.40 9 0.0047 371
    75 15 ENV 15 NLSVPNPL L L 8 372
    90 18 POL 411 NLSWLSLDV L V 927.42 9 * 0.0650 0.0051 0.6400 0.1600 0.0990 373
    90 18 POL 411 NLSWLSLDVSA L A 11 374
    100 20 POL 47 NVSIPWTHKV V V 1.0532 10 0.0001 375
    100 20 POL 430 PAAMPHLL A L 8 376
    85 17 POL 430 PAAMPHLLV A V 9 377
    90 18 POL 775 PADDPSRGRL A L 10 378
    90 18 ENV 131 PAGGSSSGT A T 9 379
    90 18 ENV 131 PAGGSSSGTV A V 10 380
    95 19 POL 641 PALMPLYA A A 8 381
    95 19 POL 641 PALMPLYACI A I 5.0087 10 0.0001 382
    75 15 X 145 PAPCNFFT A T 8 383
    75 15 X 145 PAPCNFFTSA A A 10 384
    80 16 X 11 PARDVLCL A L 8 385
    75 15 X 11 PARDVLCLRPV A V 11 386
    90 18 POL 355 PARVTTGGV A V 8 387
    90 18 POL 355 PARVTGGVFL A L 10 388
    90 18 POL 355 PARVTGGVFLV A V 11 389
    95 19 NUC 130 PAYRPPNA A A 8 390
    95 19 NUC 130 PAYRPPNAPI A I 5.0081 10 0.0001 391
    95 19 NUC 130 PAYRPPNAPIL A L 11 392
    85 17 POL 616 PIDWKVCQRI I I Chisari 10 0.0001 393
    4.091
    85 17 POL 616 PIDWKVCQRIV I V 11 394
    100 20 ENV 380 PIFFCLWV I V 8 395
    100 20 ENV 380 PIFFCLWVYI I I Chisari 10 0.0004 396
    3.074
    85 17 POL 713 PIHTAELL I L 8 397
    85 17 POL 713 PIHTAELLA I A 9 398
    85 17 POL 713 PIHTAELLAA I A 10 399
    80 16 POL 496 PIILGFRKI I I 927.48 9 0.0001 400
    80 16 POL 496 PIILGFRKIPM I M 11 401
    100 20 NUC 138 PILSTLPET I T 5.0023 9 0.0001 402
    100 20 NUC 138 PILSTLPETT I T 5.0082 10 0.0001 403
    100 20 NUC 138 PILSTLPETTV I V Chisari 11 0.0001 404
    5.125
    80 16 ENV 314 PIPSSWAFA I A 9 405
    95 19 POL 20 PLEEELPRL L L 927.29 9 0.0003 406
    90 18 POL 20 PLEEELPRLA L A 3.0225 10 0.0001 407
    95 19 ENV 10 PLGFFPDHQL L L 1.0511 10 0.0002 408
    100 20 POL 427 PLHPAAMPHL L L 1.0560 10 0.0001 409
    100 20 POL 427 PLHPAAMPHLL L L 11 410
    100 20 ENV 377 PLLPIFFCL L L 1069.13 9 * 0.0650 0.0001 0.0018 0.1100 0.0047 411
    100 20 ENV 377 PLLPIFFCLWV L V 11 412
    90 18 ENV 174 PLLVLQAGFFL L L Chisari 11 0.0008 413
    4.029
    80 16 POL 711 PLPIHTAEL L L 927.19 9 0.0004 414
    80 16 POL 711 PLPIHTAELL L L 1.0569 10 0.0001 415
    80 16 POL 711 PLPIHTAELLA L A 11 416
    75 15 POL 2 PLSYQHFRKL L L 1.0527 10 0.0001 417
    75 15 POL 2 PLSYQHFRKLL L L 11 418
    85 17 POL 98 PLTVNEKRRL L L 1.0536 10 0.0001 419
    80 16 POL 505 PMGVGLSPFL M L 1.0557 10 0.0001 420
    80 16 POL 505 PMGVGLSPFLL M L 11 421
    75 15 POL 692 PTGWGLAI T I 8 422
    80 16 ENV 219 PTSNHSPT T T 8 423
    85 17 POL 797 PTTGRTSL T L 8 424
    85 17 POL 797 PTTGRTSLYA T A 10 425
    80 16 NUC 15 PTVQASKL T L 8 428
    80 16 NUC 15 PTVQASKLCL T L 10 427
    75 15 ENV 351 PTVWLSVI T I 8 428
    75 15 ENV 351 PTVWLSVIWM T M 10 429
    95 19 X 59 PVCAFSSA V A 8 430
    85 17 POL 612 PVNRPIDWKV V V 1.0565 10 0.0002 431
    95 19 POL 654 QAFTFSPT A T 8 432
    95 19 POL 654 QAFTFSPTYKA A A 11 433
    95 19 ENV 179 QAGFFLLT A T 8 434
    80 16 ENV 179 QAGFFLLTRI A I 10 435
    80 16 ENV 179 QAGFFLLTRIL A L 11 436
    90 18 NUC 57 QAILCWGEL A L 9 437
    90 18 NUC 57 QAILCWGELM A M 10 438
    95 19 ENV 107 OAMQWNST A T 8 439
    80 16 ENV 107 QAMQWNSTT A T 9 440
    80 16 NUC 18 QASKLCLGWL A L 10 441
    80 16 X 8 QLDPARDV L V Chisari 8 0.0001 442
    4.116
    80 16 X 8 QLDPARDVL L L 927.01 9 0.0001 443
    80 16 X 8 QLDPARDVLCL L L Chisari 11 0.0001 444
    4.073
    90 18 NUC 99 QLLWFHISCL L L 1142.03 10 * 0.0060 445
    85 17 NUC 99 QLLWFHISCLT L T 11 446
    95 19 POL 685 QVFADATPT V T 5.0038 9 0.0001 447
    95 19 POL 528 RAFRHCLA A A 8 448
    80 16 ENV 187 RILTIPQSL I L Chisari 9 0.0010 449
    4.054
    90 18 POL 624 RIVGLLGFA I A 9 450
    90 18 POL 624 RIVGLLGFAA I A 10 451
    75 15 POL 106 RLKLIMPA L A 8 452
    90 18 POL 353 RTPARVTGGV T V 10 453
    95 19 NUC 127 RTPPAYRPPNA T A 11 454
    95 19 POL 36 RVAEDLNL V L 8 455
    90 18 POL 36 RVAEDLNLGNL V L 11 456
    80 16 POL 818 RVHFASPL V L 8 457
    75 15 POL 818 RVHFASPLHV V V 1.0576 10 0.0001 458
    75 15 POL 818 RVHFASPLHVA V A 11 459
    100 20 POL 357 RVTGGVFL V L 8 460
    100 20 POL 357 RVTGGVFLV V V 1.0181 9 0.0041 461
    90 18 X 65 SAGPCALRFT A T 10 462
    95 19 POL 520 SAICSVVRRA A A 5.0088 10 0.0001 463
    90 18 NUC 35 SALYREAL A L 8 464
    100 20 POL 49 SIPWTHKV I V 8 465
    95 19 ENV 194 SLDSWWTSL L L F126.64 9 466
    75 15 POL 565 SLGIHLNPNKT L T 11 467
    95 19 ENV 337 SLLVPFVQWFV L V 11 468
    75 15 POL 581 SLNFMGYV L V 8 469
    75 15 POL 581 SLNFMGYVI L I 927.12 9 0.0038 470
    95 19 X 54 SLRGLPVCA L A 3.0030 9 0.0007 471
    90 18 POL 403 SLTNLLSSNL L L 1.0548 10 0.0014 472
    75 15 ENV 280 STGPCKTCT T T 9 473
    100 20 NUC 141 STLPETTV T V 8 474
    100 20 NUC 141 STLPETTVV T V 5.0024 9 0.0019 475
    80 16 ENV 85 STNRQSGRQPT T T 11 476
    85 17 POL 548 SVQHLESL V L 8 477
    80 16 ENV 330 SVRFSWLSL V L Chisari 9 0.0001 478
    4.025
    80 16 ENV 330 SVRFSWLSLL V L Chisari 10 0.0004 479
    4.026
    80 16 ENV 330 SVRFSWLSLLV V V 11 480
    90 18 POL 739 SVVLSRKYT V T 9 481
    95 19 POL 524 SVVRRAFPHCL V L 11 482
    85 17 POL 716 TAELLAACFA A A 10 483
    95 19 NUC 53 TALRQAIL A L 8 484
    80 16 NUC 33 TASALYREA A A 9 485
    80 16 NUC 33 TASALYREAL A L 10 486
    90 18 ENV 190 TIPQSLDSWWT I T 11 487
    100 20 NUC 142 TLPETTVV L V 8 488
    100 20 POL 150 TLWKAGIL L L 8 489
    85 17 POL 798 TTGRTSLYA T A 9 490
    75 15 ENV 278 TTSTGPCKT T T 9 491
    75 15 ENV 278 TTSTGPCKTCT T T 11 492
    85 17 POL 100 TVNEKRRL V L 8 493
    80 16 NUC 16 TVQASKLCL V L 1.0365 9 0.0002 494
    75 15 ENV 352 TVWLSVIWM V M 3.0035 9 0.0002 495
    95 19 POL 37 VAEDLNLGNL A L 5.0089 10 0.0001 496
    95 19 X 15 VLCLRPVGA L A 3.0028 9 0.0014 497
    85 17 POL 543 VLGAKSVQHL L L 1.0560 10 0.0001 498
    90 18 X 133 VLGGCRHKL L L 927.08 9 0.0009 499
    90 18 X 133 VLGGCRHKLV L V 1.0589 10 0.0001 500
    85 17 X 92 VLHKRTLGL L L 927.03 9 0.0012 501
    95 19 ENV 259 VLLDYQGM L M 17.0107 8 502
    95 19 ENV 259 VLLDYQGML L L 1069.09 9 * 0.0440 0.0001 0.0210 0.9000 0.0002 503
    90 18 ENV 259 VLLDYQGMLPV L V 1147.14 11 * 0.5800 0.2200 4.9000 0.3400 0.0170 504
    95 19 ENV 177 VLQAGFFL L L Chisari 6 0.0019 505
    4.027
    95 19 ENV 177 VLQAGFFLL L L 1013.14 9 * 0.0660 506
    95 19 ENV 177 VLQAGFFLLT L T 5.0066 10 0.0011 507
    100 20 POL 358 VTGGVFLV T V 8 508
    90 18 POL 542 VVLGAKSV V V 8 509
    80 16 POL 542 VVLGAKSVQHL V L 11 510
    90 18 POL 740 VVLSRKYT V T 8 511
    95 19 POL 525 VVRRAFPHCL V L 2.0217 10 0.0003 512
    95 19 POL 525 VVRRAFPHCLA V A 11 513
    80 16 POL 759 WILRGTSFV I V 927.24 9 * 0.0270 514
    80 16 POL 759 WILRGTSFVYV I V 11 515
    80 16 POL 751 WLLGCAANWI L I Chisari 10 0.0053 516
    4.104
    80 16 POL 751 WLLGCAANWIL L L 11 517
    100 20 POL 414 WLSLDVSA L A 8 518
    95 19 POL 414 WLSLDVSAA L A 3.0023 9 0.0059 519
    100 20 ENV 335 WLSLLVPFV L V 1013.0102 9 * 1.1000 0,0380 7.2000 0.3600 0.0310 520
    95 19 ENV 237 WMCLRRFI M I 8 521
    95 19 ENV 237 WMCLRRFII M I 1147.10 9 * 0.0005 522
    95 19 ENV 237 WMCLRRFIIFL M L Chisari 11 0.0019 523
    4.024
    85 17 ENV 359 WMMWYWGPSL M L 1137.05 10 * 0.0009 524
    100 20 POL 52 WTHKVGNFT T T 5.0039 9 0.0001 525
    95 19 POL 52 WTHKVGNFTGL T L 11 526
    100 20 POL 147 YLHTLWKA L A 8 527
    100 20 POL 147 YLHTLWKAGI L I 1069.11 10 * 0.0160 0.0005 0.5600 0.1000 0.0320 528
    100 20 POL 147 YLHTLWKAGIL L L 11 529
    100 20 POL 122 YLPLDKGI L I 8 530
    90 18 NUC 118 YLVSFGVWI L I 1090.12 9 * 0.3800 531
    90 18 NUC 118 YLVSFGVWIRT L T 11 532
    90 18 POL 538 YMDDVVLGA M A 1090.14 9 * 0.0250 0.0001 0.0024 0.1000 0.0002 533
    85 17 POL 746 YTSFPWLL T L 8 534
    75 15 POL 746 YTSFPWLLGCA T A 11 535
    90 18 POL 768 YVPSALNPA V A 3.0042 9 0.0039 536
    388
  • TABLE VIII
    HBV A03 SUPER MOTIF (With binding Information)
    EQ
    Conser- Fre- Posi- C- ID
    vancy quency Protein tion Sequence P2 term Peptide AA Filed A*0201 A*0202 A*0203 A*0206 A*6802 NO:
    65 17 POL 721 AACFARSR A R 26.0003 8 0.0004 0.0003 0.0056 0.0035 0.0014 537
    95 19 POL 521 AICSVVRR I R 26.0004 8 −0.0002 0.0003 0.0014 −0.0009 0.0006 538
    90 18 POL 772 ALNPADDPSR L R 1.1090 10 0.0003 0.0001 539
    85 17 X 70 ALRFTSAR L R 26.0005 8 0.0047 0.0009 0.0450 0.0230 0.0004 540
    80 16 POL 822 ASPLHVAWR S R 9 641
    75 15 ENV 84 ASTNRQSGR S R 1150.60 9 0.0009 0.0002 0.0088 0.0008 0.0001 542
    80 16 POL 755 CAANWILR A R 8 543
    85 17 X 69 CALRFTSAR A R 26.0149 9 * 0.0034 0.0230 1.5000 8.0000 0.7300 644
    90 18 X 17 CLRPVGAESR L R 1.1093 10 0.0011 0.0001 545
    100 20 NUC 48 CSPHHTALR S R 5.0055 9 * 0.0029 0.0001 0.0520 0.0250 0.0440 546
    85 17 NUC 29 DLLDTASALYR L R 26.0530 11 0.0042 −0.0003 −0.0012 3.7000 0.0410 547
    85 17 NUC 32 DTASALYR T R 26.0006 8 0.0004 −0.0002 −0.0009 0.0018 0.0009 548
    95 19 POL 17 EAGPLEEELPR A R 26.0531 11 −0.0009 −0.0003 −0.0012 0.0015 0.0110 549
    90 18 POL 718 ELLAACFAR L R 1.0988 9 0.0002 0.0004 550
    65 17 POL 718 ELLAACFARSR L R 26.0532 11 0.0062 0.0016 0.0200 0.2000 0.1600 551
    95 19 NUC 174 ETTVVRRR T R 26.0007 8 0.0003 −0.0002 −0.0009 0.1400 0.0027 552
    80 16 NUC 174 ETTVVRRRGR T R 1.1073 10 0.0003 0.0001 553
    80 16 POL 821 FASPLHVAWR A R 10 554
    90 18 X 63 FSSAGPCALR S R 10 555
    95 19 POL 656 FTFSPTYK T K 1147.19 8 * 0.0100 0.0100 0.0023 0.2100 0.0590 556
    95 19 POL 518 FTSAICSVVR T R 1.1085 10 0.0003 0.0003 557
    95 19 POL 518 FTSAICSVVRR T R 26.0533 11 0.0065 0.0092 0.0170 0.0350 1.5000 558
    90 18 X 132 FVLGGCRHK V K 1090.03 9 * 0.0430 0.0090 559
    75 15 POL 567 GIHLNPNK I K 8 560
    75 15 POL 567 GIHLNPNKTK I K 1.0563 10 0.0025 0.0011 0.0009 0.0009 0.0003 561
    75 15 POL 567 GIHLNPNKTKR I R 11 562
    85 17 NUC 29 GMDIDPYK M K 26.0009 8 0.0006 0.0004 −0.0009 −0.0009 0.0001 563
    90 18 POL 735 GTDNSVVLSR T R 1090.04 10 * 0.0010 0.0420 0.0030 0.0019 0.0008 564
    90 18 POL 735 GTDNSVVLSRK T K 1147.17 11 * 0.0140 0.5600 −0.0002 −0.0006 0.0001 565
    95 19 NUC 123 GVWIRTPPAYR V R 26.0535 11 * 0.1900 0.1700 6.8000 0.7300 0.6600 566
    90 18 NUC 104 HISCLTFGR I R 1069.18 9 * 0.0160 0.0065 567
    75 15 POL 569 HLNPNKTK L K 8 568
    75 15 POL 569 HLNPNKTKR L R 1.0983 9 * 0.0025 0.0001 569
    100 20 POL 149 HTLWKAGILYK T K 1147.16 11 * 0.5400 0.4400 0.0370 0.0720 0.1900 570
    90 18 NUC 105 ISCLTFGR S R 26.0010 8 0.0004 0.0002 0.0017 −0.0009 0.0017 571
    100 20 POL 153 KAGILYKR A R 26.0011 8 0.0002 −0.0002 0.0015 −0.0009 0.0001 572
    80 16 POL 610 KLPVNRPIDWK L K 11 573
    75 15 X 130 KVFVLGGCR V R 1.0993 9 * 0.0420 0.0620 0.6000 0.0710 0.0030 574
    85 17 POL 720 LAACFARSR A R 20.0129 9 * 0.0058 0.0065 575
    90 18 POL 719 LLAACFAR L R 26.0012 8 0.0024 0.0003 0.0015 0.0029 0.0064 576
    85 17 POL 719 LLAACFARSR L R 10 577
    85 17 NUC 30 LLDTASALYR L R 1.1070 10 0.0050 0.0002 578
    80 16 POL 752 LLGCAANWILR L R 11 579
    75 15 POL 564 LSLGIHLNPNK S K 11 580
    95 19 NUC 169 LSTLPETTVVR S R 26.0537 11 −0.0009 0.0008 −0.0012 −0.0023 0.0078 581
    75 15 POL 3 LSYQHFRK S K 8 582
    85 17 POL 99 LTVNEKRR T R 26.0013 8 −0.0002 −0.0002 −0.0009 −0.0009 0.0001 583
    90 18 NUC 119 LVSFGVWIR V R 1090.08 9 * 0.0028 0.0120 584
    100 20 POL 377 LVVDFSQFSR V R 1069.20 10 * 0.0016 0.3600 0.0260 0.2300 0.4900 585
    75 15 X 103 MSTTDLEAYFK S K 11 586
    90 18 NUC 75 NLEDPASR L R 26.0014 8 −0.0002 −0.0002 −0.0009 −0.0009 0.0001 587
    95 19 POL 45 NLNVSIPWTHK L K 26.0538 11 −0.0009 0.0005 −0.0012 −0.0023 0.0019 588
    90 18 POL 738 NSVVLSRK S K 26.0015 8 0.0006 0.0010 −0.0009 −0.0009 0.0007 589
    100 20 POL 47 NVSIPWTHK V K 1069.16 9 * 0.0820 0.0570 0.0002 0.0100 0.0320 590
    90 18 POL 775 PADDPSRGR A R 1150.35 9 0.0008 0.0002 0.0004 0.0015 0.0002 591
    80 16 X 11 PARDVLCLR A R 1150.36 9 0.0002 0.0002 0.0100 0.0180 0.0002 592
    75 15 ENV 83 PASTNRQSGR A R 10 593
    90 18 POL 616 PIDWKVCQR I R 1.0985 9 0.0002 0.0005 594
    80 16 POL 496 PIILGFRK I K 8 595
    95 19 POL 20 PLEEELPR L R 26.0016 8 0.0002 −0.0002 −0.0009 −0.0009 0.0001 596
    100 20 POL 2 PLSYQHFR L R 26.0017 8 −0.0002 −0.0002 −0.0009 −0.0009 0.0001 597
    75 15 POL 2 PLSYQHFRK L K 1.0161 9 0.0011 0.0031 0.0006 0.0008 0.0002 598
    85 17 POL 98 PLTVNEKR L R 26.0018 8 0.0002 −0.0002 −0.0009 −0.0009 0.0001 599
    85 17 POL 98 PLTVNEKRR L R 1.0974 9 0.0008 0.0005 0.0004 0.0027 0.0002 600
    90 18 X 20 PVGAESRGR V R 1.0990 9 0.0002 0.0005 0.0004 0.0043 0.0002 601
    85 17 POL 612 PVNRPIDWK V K 1142.06 9 * 0.0310 0.1400 0.0002 0.0006 0.0009 602
    95 19 POL 654 QAFTFSPTYK A K 1090.10 10 * 0.0450 0.5400 0.0010 0.0057 1.2000 603
    80 16 ENV 179 QAGFFLLTR A R 9 604
    75 15 NUC 169 QSPRRRRSQSR S R 28.0839 11 605
    80 16 POL 189 QSSGILSR S R 8 606
    75 15 POL 106 RLKLIMPAR L R 1.0975 9 * 0.0950 0.0002 3.1000 0.0490 0.0002 607
    75 15 X 128 RLKVFVLGGCR L R 11 608
    95 19 POL 376 RLVVDFSQFSR L R 26.0639 1 * 0.2800 3.8000 2.6000 1.2000 6.1000 609
    95 19 NUC 183 RSPRRRTPSPR S R 26.0540 11 −0.0007 −0.0003 0.0190 −0.0023 0.0003 610
    75 15 NUC 167 RSQSPRRR S R 8 611
    75 15 NUC 167 RSQSPRRRR S R 9 612
    95 19 NUC 188 RTPSPRRR T R 26.0019 8 −0.0002 −0.0002 0.0033 0.0014 0.0002 613
    95 19 NUC 188 RTPSPRRRR T R 1.0971 9 * 0.0054 0.0005 0.2000 0.0016 0.0003 614
    100 20 POL 357 RVTGGVFLVDK V K 1147.18 11 * 0.0190 0.0290 −0.0002 −0.0003 0.0001 615
    90 18 X 65 SAGPCALR A R 26.0020 8 −0.0002 0.0020 0.0029 0.0024 0.0360 616
    95 19 POL 520 SAICSVVR A R 26.0021 8 −0.0002 0.0071 0.0280 0.0081 0.0690 617
    95 19 POL 520 SAICSVVRR A R 1090.11 9 * 0.0058 0.2100 0.1500 0.0650 0.3800 618
    90 18 POL 771 SALNPADDPSR A R 26.0542 11 −0.0004 −0.0003 −0.0012 −0.0023 0.0003 619
    75 15 POL 565 SLGIHLNPNK L K 28.0758 10 * 620
    90 18 X 64 SSAGPCALR S R 26.0153 9 * 0.0080 0.1400 0.3300 0.1600 0.7500 621
    95 19 NUC 170 STLPETTVVR T R 1069.21 10 * 0.0007 0.0600 0.0080 0.0240 0.0250 622
    95 19 NUC 170 STLPETTVVRR T R 1083.01 11 0.0150 1.4000 0.1000 0.1600 0.3100 623
    80 16 ENV 85 STNRQSGR T R 8 624
    75 15 X 104 STTDLEAYFK T K 1.0584 10 * 0.0066 2.7000 625
    85 17 POL 716 TAELLAACFAR A R 26.0544 11 0.0006 0.0023 0.0066 0.1600 0.0590 626
    95 19 NUC 171 TLPETTVVR L R 1.0969 9 0.0008 0.0002 0.0009 0.0024 0.0180 627
    95 19 NUC 171 TLPETTVVRR L R 1069.22 10 * 0.0007 0.0230 0.0006 0.0120 0.0440 628
    95 19 NUC 171 TLPETTVVRRR L R 26.0545 11 * 0.0005 0.0160 0.0061 0.0710 0.6400 629
    100 20 POL 150 TLWKAGILYK L K 1069.15 10 * 5.3000 0.3600 0.0051 0.0010 0.0130 630
    100 20 POL 150 TLWKAGILYKR L R 26.0546 11 0.0082 0.0095 0.1000 0.1100 0.0640 631
    95 19 POL 519 TSAICSVVR S R 5.0057 9 0.0005 0.0008 0.0600 0.0200 0.0820 632
    95 19 POL 519 TSAICSVVRR S R 1142.08 10 * 0.0018 0.0006 0.0030 0.0066 0.0048 633
    75 15 X 105 TTDLEAYFK T K 1.0215 9 * 0.0006 0.9200 0.0006 0.0012 0.0170 634
    75 15 ENV 278 TTSTGPCK T K 8 635
    80 16 NUC 175 TTVVRRRGR T R 1.0970 9 0.0008 0.0005 0.2500 0.1400 0.0095 636
    80 16 NUC 176 TVVRRRGR V R 3.0324 8 0.0003 0.0001 637
    80 16 NUC 176 TVVRRRGRSPR V R 28.0837 11 638
    90 18 X 133 VLGGCRHK L K 26.0022 8 0.0150 0.0002 −0.0005 −0.0009 0.0001 639
    80 16 ENV 177 VLQAGFFLLTR L R 11 640
    90 18 NUC 120 VSFGVWIR S R 26.0023 8 * 0.0040 0.0290 0.0750 0.0270 0.0360 641
    100 20 POL 48 VSIPWTHK S K 26.0024 8 * 0.0130 0.0170 0.0031 0.0013 0.0004 642
    100 20 POL 358 VTGGVFLYDK T K 1069.17 10 * 0.0390 0.0920 0.0002 0.0006 0.0022 643
    100 20 POL 378 VVDFSQFSR V R 1069.19 9 * 0.0015 0.0750 0.0013 0.0170 0.0330 644
    80 16 NUC 177 VVRRRGRSPR V R 1.1074 10 0.0027 0.0001 645
    80 16 NUC 177 VVRRRGRSPRR V R 28.0838 11 646
    95 19 NUC 125 WIRTPPAYR I R 1.0968 9 0.0008 0.0005 647
    90 18 POL 314 WLQFRNSK L K 26.0025 8 −0.0002 0.0005 0.0020 0.0052 0.0001 648
    85 17 NUC 26 WLWGMDIDPYK L K 26.0547 11 0.0030 0.0013 −0.0003 0.0039 0.0490 649
    100 20 POL 122 YLPLDKGIK L K 1.0173 9 0.0001 0.0001 0.0006 0.0006 0.0002 650
    90 18 NUC 118 YLVSFGVWIR L R 1090.13 10 * 0.0005 0.0002 651
    90 18 POL 538 YMDDVVLGAK M K 1090.15 10 * 0.0330 0.0043 0.0002 0.0006 0.0001 652
    80 16 POL 493 YSHPIILGFR S R 10 653
    80 16 POL 493 YSHPIILGFRK S K 11 654
    118
  • TABLE IX
    HBV A24 SUPER MOTIF (With binding information)
    SEQ ID
    Conservancy Freq Protein Position Sequence String Peptide Filed A*2401 NO
    95 19 POL 529 AFPHCLAF XFXXXXXF 655
    95 19 POL 529 AFPHCLAFSY XFXXXXXXXY 656
    95 19 POL 529 AFPHCLAFSYM XFXXXXXXXXM 657
    95 19 X 62 AFSSAGPCAL XFXXXXXXXL 5.0118 0.0012 658
    90 18 POL 535 AFSYMDDVVL XFXXXXXXXL 13.0130 0.0009 659
    95 19 POL 655 AFTFSPTY XFXXXXXY 660
    95 19 POL 655 AFTFSPTYKAF XFXXXXXXXXF 661
    95 19 POL 521 AICSVVRRAF XIXXXXXXXF 662
    90 18 NUC 58 AILCWGEL XIXXXXXL 663
    90 18 NUC 58 AILCWGELM XIXXXXXXM 664
    95 19 POL 642 ALMPLYACI XLXXXXXXI 3.0012 * 665
    95 19 NUC 54 ALRQAILCW XLXXXXXXW 666
    80 16 ENV 108 AMQWNSTTF XMXXXXXXF 667
    95 19 POL 690 ATPTGWGL XTXXXXXL 668
    75 15 POL 690 ATPTGWGLAI XTXXXXXXXI 669
    95 19 POL 397 AVPNLQSL XVXXXXXL 670
    95 19 POL 397 AVPNLQSLTNL XVXXXXXXXXL 671
    100 20 NUC 131 AYRPPNAPI XYXXXXXXI 5.0082 * 0.0260 672
    100 20 NUC 131 AYRPPNAPIL XYXXXXXXXL 2.0172 * 0.0220 673
    75 15 POL 607 CFRKLPVNRPI XFXXXXXXXXI 674
    100 20 ENV 312 CIPIPSSW XIXXXXXW 675
    100 20 ENV 312 CIPIPSSWAF XIXXXXXXXF 676
    85 17 NUC 23 CLGWLWGM XLXXXXXM 677
    85 17 NUC 23 CLGWLWGMDI XLXXXXXXXI 2.0229 678
    100 20 ENV 253 CLIFLLVL XLXXXXXL 17.0248 679
    100 20 ENV 253 CLIFLLVLL XLXXXXXXL 1.0836 680
    95 19 ENV 253 CLIFLLVLLDY XLXXXXXXXXY 26.0548 681
    95 19 ENV 239 CLRRFIIF XLXXXXXF 682
    95 19 ENV 239 CLRRFIIFL XLXXXXXXL 1.0829 683
    75 15 ENV 239 CLRRFIIFLF XLXXXXXXXF 684
    75 15 ENV 239 CLRRFIIFLFI XLXXXXXXXXI Chisari 685
    4.055
    100 20 ENV 310 CTCIPIPSSW XTXXXXXXXW 686
    90 18 NUC 31 DIDPYKEF XIXXXXXF 687
    85 17 NUC 29 DLLDTASAL XLXXXXXXL 1.0154 688
    85 17 NUC 29 DLLDTASALY XLXXXXXXXY 1.0519 * 689
    95 19 POL 40 DLNLGNLNVSI XLXXXXXXXXI 690
    80 16 NUC 32 DTASALYREAL XTXXXXXXXXL 691
    85 17 POL 618 DWKVCQRI XWXXXXXI 692
    85 17 POL 618 DWKVCQRIVGL XWXXXXXXXXL 693
    90 18 ENV 262 DYQGMLPVCPL XYXXXXXXXXL 3.0441 0.0002 694
    80 16 X 122 ELGEEIRL XLXXXXXL 695
    95 19 NUC 43 ELLSFLPSDF XLXXXXXXXF 696
    95 19 NUC 43 ELLSFLPSDFF XLXXXXXXXXF 697
    90 18 NUC 117 EYLVSFGVW XYXXXXXXW 26.0150 698
    90 18 NUC 117 EYLVSFGVWI XYXXXXXXXI 13.0129 * 0.0340 699
    100 20 ENV 382 FFCLWVYI XFXXXXXI 700
    80 16 ENV 182 FFLLTRIL XFXXXXXL 701
    80 16 ENV 182 FFLLTRILTI XFXXXXXXXI 702
    85 17 ENV 13 FFPDHQLDPAF XFXXXXXXXXF 703
    80 16 ENV 243 FIIFLFIL XIXXXXXL 17.0246 704
    80 16 ENV 243 FIIFLFILL XIXXXXXXL 1.0830 705
    80 16 ENV 243 FIIFLFILLL XIXXXXXXXL 1.0694 706
    80 16 ENV 248 FILLLCLI XIXXXXXI Chisari 707
    4.045
    80 16 ENV 248 FILLLCLIF XIXXXXXXF 708
    80 16 ENV 248 FILLLCLIFL XIXXXXXXXL 1.0895 * 709
    80 16 ENV 248 FILLLCLIFLL XIXXXXXXXXL Chisari 710
    4.049
    80 16 ENV 246 FLFILLLCL XLXXXXXXL 1.0832 711
    80 16 ENV 246 FLFILLLCLI XLXXXXXXXI 3.0206 * 712
    80 16 ENV 246 FLFILLLCLIF XLXXXXXXXXF 713
    75 15 ENV 171 FLGPLLVL XLXXXXXL 714
    95 19 POL 513 FLLAQFTSAI XLXXXXXXXI 1147.13 * 715
    95 19 POL 562 FLLSLGIHL XLXXXXXXL 1.0851 * 716
    80 16 ENV 183 FLLTRILTI XLXXXXXXI 3.0005 * 717
    95 19 ENV 256 FLLVLLDY XLXXXXXY 26.0027 718
    95 19 ENV 256 FLLVLLDYQGM XLXXXXXXXXM 719
    95 19 POL 656 FTFSPTYKAF XTXXXXXXXF 20.0262 720
    95 19 POL 656 FTFSPTYKAFL XTXXXXXXXXL 721
    95 19 POL 635 FTQCGYPAL XTXXXXXXL 5.0031 722
    95 19 POL 635 FTQCGYPALM XTXXXXXXXM 5.0085 723
    95 19 ENV 346 FVGLSPTVW XVXXXXXXW 724
    95 19 ENV 346 FVGLSPTVWL XVXXXXXXXL 1.0931 725
    90 18 X 132 FVLGGCRHKL XVXXXXXXXL 1.0588 726
    95 19 ENV 342 FVQWFVGL XVXXXXXL 17.019 * 727
    90 18 POL 766 FVYVPSAL XVXXXXXL 17.0260 * 728
    95 19 POL 630 GFAAPFTQCGY XFXXXXXXXXY 729
    80 16 ENV 181 GFFLLTRI XFXXXXXI 730
    80 16 ENV 181 GFFLLTRIL XFXXXXXXL 731
    80 16 ENV 181 GFFLLTRILTI XFXXXXXXXXI 732
    95 19 ENV 12 GFFPDHQL XFXXXXXL 733
    75 15 ENV 170 GFLGPLLVL XFXXXXXXL 734
    80 16 POL 500 GFRKIPMGVGL XFXXXXXXXXL 735
    95 19 POL 627 GLLGFAAPF XLXXXXXXF 20.0124 736
    95 19 POL 509 GLSPFLLAQF XLXXXXXXXF 737
    100 20 ENV 348 GLSPTVWL XLXXXXXL Chisari 738
    4.012
    75 15 ENV 348 GLSPTVWLSVI XLXXXXXXXXI Chisari 739
    4.031
    85 17 NUC 29 GMDIDPYKEF XMXXXXXXXF 26.0372 740
    90 18 ENV 265 GMLPVCPL XMXXXXXL 741
    90 18 POL 735 GTDNSVVL XTXXXXXL 742
    75 15 ENV 13 GTNLSVPNPL XTXXXXXXXL 743
    80 16 POL 763 GTSFVYVPSAL XTXXXXXXXXL 744
    80 16 POL 507 GVGLSPFL XVXXXXXL 745
    80 16 POL 507 GVGLSPFLL XVXXXXXXL 1.0850 746
    95 19 NUC 128 GVWIRTPPAY XVXXXXXXXY 1.0525 747
    85 17 NUC 25 GWLWGMDI XWXXXXXI 748
    85 17 NUC 25 GWLWGMDIDPY XWXXXXXXXXY 749
    85 17 ENV 65 GWSPQAQGI XWXXXXXXI 20.0134 0.0024 750
    85 17 ENV 65 GWSPQAQGIL XWXXXXXXXL 20.0268 0.0003 751
    95 19 POL 639 GYPALMPL XYXXXXXL 752
    95 19 POL 639 GYPALMPLY XYXXXXXXY 2.0060 * 0.0490 753
    95 19 ENV 234 GYRWMCLRRF XYXXXXXXXF 2.0171 * 0.0110 754
    95 19 ENV 234 GYRWMCLRRFI XYXXXXXXXXI 755
    85 17 POL 579 GYSLNFMGY XYXXXXXXY 2.0058 0.0002 756
    75 15 POL 579 GYSLNFMGYVI XYXXXXXXXXI 757
    80 16 POL 820 HFASPLHVAW XFXXXXXXXW 758
    75 15 POL 7 HFRKLLLL XFXXXXXL 759
    80 16 POL 435 HLLVGSSGL XLXXXXXXL 1.0187 760
    75 15 POL 569 HLNPNKTKRW XLXXXXXXXW 761
    80 16 POL 491 HLYSHPII XLXXXXXI 17.0256 762
    80 16 POL 491 HLYSHPIIL XLXXXXXXL 1.0849 * 763
    80 16 POL 491 HLYSHPIILGF XLXXXXXXXXF 764
    85 17 POL 715 HTAELLAACF XTXXXXXXXF 765
    100 20 NUC 52 HTALRQAI XTXXXXXI 766
    95 19 NUC 52 HTALAQAIL XTXXXXXXL 5.0021 767
    95 19 NUC 52 HTALRQAILCW XTXXXXXXXXW 768
    100 20 POL 149 HTLWKAGI XTXXXXXI 769
    100 20 POL 149 HTLWKAGIL XTXXXXXXL 5.0033 770
    100 20 POL 149 HTLWKAGILY XTXXXXXXXY 1.0542 * 771
    100 20 POL 146 HYLHTLWKAGI XYXXXXXXXXI 772
    100 20 ENV 381 IFFCLWVY XFXXXXXY 773
    100 20 ENV 381 IFFCLWVYI XFXXXXXXI 5.0058 0.0087 774
    80 16 ENV 245 IFLFILLL XFXXXXXL 775
    80 16 ENV 245 IFLFILLLCL XFXXXXXXXL 776
    80 16 ENV 245 IFLFILLLCLI XFXXXXXXXXI 777
    95 19 ENV 255 IFLLVLLDY XFXXXXXXY 778
    80 16 ENV 244 IIFLFILL XIXXXXXL 17.0105 779
    80 16 ENV 244 IIFLFILLL XIXXXXXXL 1.0831 780
    80 16 ENV 244 IIFLFILLLCL XIXXXXXXXXL Chisari 781
    4.052
    80 16 POL 497 IILGFRKI XIXXXXXI 17.0124 * 782
    80 16 POL 497 IILGFRKIPM XIXXXXXXXM 783
    90 18 NUC 59 ILCWGELM XLXXXXXM 784
    80 16 POL 498 ILGFRKIPM XLXXXXXXM 3.0016 785
    100 20 ENV 249 ILLLCLIF XLXXXXXF 786
    100 20 ENV 249 ILLLCLIFL XLXXXXXXL 1.0833 * 787
    100 20 ENV 249 ILLLCLIFLL XLXXXXXXXL 1.0896 * 788
    80 16 POL 760 ILRGTSFVY XLXXXXXXY 1.0205 * 789
    95 19 ENV 188 ILTIPQSL XLXXXXXL 790
    90 18 ENV 188 ILTIPQSLDSW XLXXXXXXXXW 791
    90 18 POL 625 IVGLLGFAAPF XVXXXXXXXXF 792
    85 17 ENV 358 IWMMWYWGPS XWXXXXXXXXL 1039.07 0.0004 793
    95 19 POL 395 KFAVPNLQSL XFXXXXXXXL 5.0114 0.0020 794
    80 16 POL 503 KIPMGVGL XIXXXXXL 795
    80 16 POL 503 KIPMGVGLSPF XIXXXXXXXXF 796
    85 17 NUC 21 KLCLGWLW XLXXXXXW 797
    85 17 NUC 21 KLCLGWLWGM XLXXXXXXXM 3.0209 * 798
    95 19 POL 489 KLHLYSHPI XLXXXXXXI 3.0009 * 799
    80 16 POL 489 KLHLYSHPII XLXXXXXXXI 800
    80 16 POL 489 KLHLYSHPIIL XLXXXXXXXXL 801
    75 15 POL 108 KLIMPARF XLXXXXXF 802
    75 15 POL 108 KLIMPARFY XLXXXXXXY 1.0171 803
    80 16 POL 610 KLPVNRPI XLXXXXXI 804
    80 16 POL 610 KLPVNRPIDW XLXXXXXXXW 805
    95 19 POL 574 KTKRWGYSL XTXXXXXXL 5.0034 806
    85 17 POL 574 KTKRWGYSLNF XTXXXXXXXXF 807
    85 17 POL 620 KVCQRIVGL XVXXXXXXL 1.0198 808
    85 17 POL 620 KVCQRIVGLL XVXXXXXXXL 1.0567 809
    95 19 POL 55 KVGNFTGL XVXXXXXL 17.0116 810
    95 19 POL 55 KVGNFTGLY XVXXXXXXY 1.0166 * 811
    85 17 X 91 KVLHKRTL XVXXXXXL 812
    85 17 X 91 KVLHKRTLGL XVXXXXXXXL 1.0800 813
    100 20 POL 121 KYLPLDKGI XYXXXXXXI 5.0063 * 0.0028 814
    85 17 POL 745 KYTSFPWL XYXXXXXL 17.0132 815
    85 17 POL 745 KYTSFPWLL XYXXXXXXL 2.0061 * 3.6000 816
    80 16 ENV 247 LFILLLCL XFXXXXXL 17.0247 817
    80 16 ENV 247 LFILLLCLI XFXXXXXXI 818
    80 16 ENV 247 LFILLLCLIF XFXXXXXXXF 819
    80 16 ENV 247 LFILLLCLIFL XFXXXXXXXXL 820
    100 20 ENV 254 LIFLLVLL XIXXXXXL Chisari 821
    4.014
    95 19 ENV 254 LIFLLVLLDY XIXXXXXXXY 1.0899 822
    100 20 POL 109 LIMPARFY XIXXXXXY 26.0028 823
    95 19 POL 514 LLAQFTSAI XLXXXXXXI 3.0010 * 824
    100 20 ENV 251 LLCLIFLL XLXXXXXL Chisari 825
    4.015
    100 20 ENV 251 LLCLIFLLVL XLXXXXXXXL 1.0898 826
    100 20 ENV 251 LLCLIFLLVLL XLXXXXXXXXL Chisari 827
    4.016
    85 17 NUC 30 LLDTASAL XLXXXXXL 828
    83 17 NUC 30 LLDTASALY XLXXXXXXY 1.0155 * 829
    95 19 ENV 260 LLDYQGML XLXXXXXL Chisari 830
    4.021
    80 16 POL 752 LLGCAANW XLXXXXXW 831
    80 16 POL 752 LLGCAANWI XLXXXXXXI 3.0013 832
    80 16 POL 752 LLGCAANWIL XLXXXXXXXL 1.0912 * 833
    95 19 POL 628 LLGFAAPF XLXXXXXF 834
    75 15 ENV 63 LLGWSPQAQGI XLXXXXXXXXI 835
    100 20 ENV 250 LLLCLIFL XLXXXXXL Chisari 836
    4.017
    100 20 ENV 250 LLLCLIFLL XLXXXXXXL 1.0834 * 837
    100 20 ENV 250 LLLCLIFLLVL XLXXXXXXXXL Chsari 838
    4.018
    100 20 ENV 378 LLPIFFCL XLXXXXXL 17.0112 839
    100 20 ENV 378 LLPIFFCLW XLXXXXXXW 840
    100 20 ENV 378 LLPIFFCLWVY XLXXXXXXXXY 26.0549 * 841
    95 19 NUC 44 LLSFLPSDF XLXXXXXXF 842
    95 19 NUC 44 LLSFLPSDFF XLXXXXXXXF 843
    95 19 POL 563 LLSLGIHL XLXXXXXL 844
    90 18 POL 407 LLSSNLSW XLXXXXXW 845
    90 18 POL 407 LLSSNLSWL XLXXXXXXL 1.0184 * 846
    90 18 POL 407 LLSSNLSWLSL XLXXXXXXXXL 847
    80 16 ENV 184 LLTRILTI XLXXXXXI Chisari 848
    4.053
    80 16 POL 436 LLVGSSGL XLXXXXXL 849
    95 19 ENV 257 LLVLLDYQGM XLXXXXXXXM 3.0207 850
    95 19 ENV 257 LLVLLDYQGML XLXXXXXXXXL 851
    95 19 ENV 175 LLVLQAGF XLXXXXXF 852
    95 19 ENV 175 LLVLQAGFF XLXXXXXXF 20.0121 853
    90 18 ENV 175 LLVLQAGFFL XLXXXXXXXL 1.0892 * 854
    90 18 ENV 175 LLVLQAGFFLL XLXXXXXXXXL Chisari 855
    4.028
    100 20 ENV 338 LLVPFVQW XLXXXXXW 556
    100 20 ENV 338 LLVPFVQWF XLXXXXXXF 857
    90 18 NUC 100 LLWFHISCL XLXXXXXXL 1.0844 * 858
    85 17 NUC 100 LLWFHISCLTF XLXXXXXXXXF 859
    95 19 POL 643 LMPLYACI XMXXXXXI 17.0130 860
    75 15 NUC 137 LTFGRETVL XTXXXXXXL 861
    75 15 NUC 137 LTFGRETVLEY XTXXXXXXXXY 862
    90 18 ENV 189 LTIPQSLDSW XTXXXXXXXW 863
    90 18 ENV 189 LTIPQSLDSWW XTXXXXXXXXW 864
    90 18 POL 404 LTNLLSSNL XTXXXXXXL 865
    90 18 POL 404 LTNLLSSNLSW XTXXXXXXXXW 866
    80 16 ENV 185 LTRILTIPQSL XTXXXXXXXXL 867
    85 17 POL 99 LTVNEKRRL XTXXXXXXL 868
    95 19 ENV 258 LVLLDYQGM XVXXXXXXM 3.0034 869
    95 19 ENV 258 LVLLDYQGML XVXXXXXXXL 1.0515 870
    95 19 ENV 176 LVLQAGFF XVXXXXXF 871
    90 18 ENV 176 LVLQAGFFL XVXXXXXXL 1.0827 872
    90 18 ENV 176 LVLQAGFFLL XVXXXXXXXL 1.0893 * 873
    100 20 ENV 339 LVPFVQWF XVXXXXXF 874
    95 19 ENV 339 LVPFVQWFVGL XVXXXXXXXXL 875
    90 18 NUC 119 LVSFGVWI XVXXXXXI Chisari 876
    4.078
    100 20 POL 377 LVVDFSQF XVXXXXXF 877
    90 18 NUC 101 LWFHISCL XWXXXXXL 878
    85 17 NUC 101 LWFHISCLTF XWXXXXXXXF 26.0373 879
    85 17 NUC 27 LWGMDIDPY XWXXXXXXY 880
    100 20 POL 151 LWKAGILY XWXXXXXY 881
    80 16 POL 492 LYSHPIIL XYXXXXXL 882
    80 16 POL 492 LYSHPIILGF XYXXXXXXXF 2.0181 * 1.1000 883
    85 17 ENV 360 MMWYWGPSL XMXXXXXXL 1.0839 * 0.0012 884
    85 17 ENV 360 MMWYWGPSLY XMXXXXXXXY 1039.01 * 0.0001 885
    85 17 ENV 361 MWYWGPSL XWXXXXXL 17.0249 886
    85 17 ENV 361 MWYWGPSLY XWXXXXXXY 1039.02 0.0027 887
    95 19 POL 561 NFLLSLGI XFXXXXXI 888
    95 19 POL 561 NFLLSLGIHL XFXXXXXXXL 5.0115 0.0099 889
    95 19 POL 42 NLGNLNVSI XLXXXXXXI 3.0008 890
    96 19 POL 42 NLGNLNVSIPW XLXXXXXXXXW 891
    90 18 POL 406 NLLSSNLSW XLXXXXXXW 892
    90 18 POL 406 NLLSSNLSWL XLXXXXXXXL 1.0549 893
    95 19 POL 45 NLNVSIPW XLXXXXXW 894
    100 20 POL 400 NLQSLTNL XLXXXXXL 895
    100 20 POL 400 NLQSLTNLL XLXXXXXXL 1.0183 896
    75 15 ENV 15 NLSVPNPL XLXXXXXL 897
    75 15 ENV 15 NLSVPNPLGF XLXXXXXXXF 898
    80 16 POL 758 NWILRGTSF XWXXXXXXF 899
    80 16 POL 758 NWILRGTSFVY XWXXXXXXXXY 900
    95 19 POL 512 PFLLAQFTSAI XFXXXXXXXXI 901
    95 19 POL 634 PFTQCGYPAL XFXXXXXXXL 5.0116 0.0002 902
    95 19 POL 634 PFTQCGYPALM XFXXXXXXXXM 903
    95 19 ENV 341 PFVQWFVGL XFXXXXXXL 5.0059 0.0003 904
    85 17 POL 616 PIDWKVCQRI XIXXXXXXXI Chisari 905
    4.091
    100 20 ENV 380 PIFFCLWVY XIXXXXXXY 1.0843 906
    100 20 ENV 380 PIFFCLWVYI XIXXXXXXXI 20.0258 907
    85 17 POL 713 PIHTAELL XIXXXXXL 908
    80 16 POL 496 PIILGFRKI XIXXXXXXI 927.48 909
    80 16 POL 496 PIILGFRKIPM XIXXXXXXXXM 910
    100 20 ENV 314 PIPSSWAF XIXXXXXF 911
    100 20 POL 124 PLDKGIKPY XLXXXXXXY 1.0174 * 912
    100 20 POL 124 PLDKGIKPYY XLXXXXXXXY 1.0541 * 913
    95 19 POL 20 PLEEELPRL XLXXXXXXL 1.0163 914
    95 19 ENV 10 PLGFFPDHQL XLXXXXXXXL 1.0511 915
    100 20 POL 427 PLHPAAMPHL XLXXXXXXXL 1.0550 916
    100 20 POL 427 PLHPAAMPHLL XLXXXXXXXXL 917
    100 20 ENV 377 PLLPIFFCL XLXXXXXXL 1.0842 * 918
    100 20 ENV 377 PLLPIFFCLW XLXXXXXXXW 919
    95 19 ENV 174 PLLVLQAGF XLXXXXXXF 920
    95 19 ENV 174 PLLVLQAGFF XLXXXXXXXF 921
    90 18 ENV 174 PLLVLQAGFFL XLXXXXXXXXL Chisari 922
    4.029
    80 16 POL 711 PLPIHTAEL XLXXXXXXL 1.0201 923
    80 16 POL 711 PLPIHTAELL XLXXXXXXXL 1.0569 924
    75 15 POL 2 PLSYQHFRKL XLXXXXXXXL 1.0527 925
    75 15 POL 2 PLSYQHFRKLL XLXXXXXXXXL 926
    85 17 POL 98 PLTVNEKRRL XLXXXXXXXL 1.0536 927
    80 16 POL 505 PMGVGLSPF XMXXXXXXF 928
    80 16 POL 505 PMGVGLSPFL XMXXXXXXXL 1.0557 929
    80 16 POL 505 PMGVGLSPFLL XMXXXXXXXXL 930
    75 15 POL 692 PTGWGLAI XTXXXXXI 931
    85 17 POL 797 PTTGRTSL XTXXXXXL 932
    85 17 POL 797 PTTGRTSLY XTXXXXXXY 1.0208 * 933
    80 16 NUC 15 PTVQASKL XTXXXXXL 934
    80 16 NUC 15 PTVQASKLCL XTXXXXXXXL 935
    75 15 ENV 351 PTVWLSVI XTXXXXXI 936
    75 15 ENV 351 PTVWLSVIW XTXXXXXXW 937
    75 15 ENV 351 PTVWLSVIWM XTXXXXXXXM 938
    85 17 POL 612 PVNRPIDW XVXXXXXW 939
    80 16 POL 750 PWLLGCAANW XWXXXXXXXW 940
    80 16 POL 750 PWLLGCAANWI XWXXXXXXXXI 941
    100 20 POL 51 PWTHKVGNF XWXXXXXXF 20.0138 * 0.0290 942
    80 16 X 8 QLDPARDVL XLXXXXXXL 1.0210 943
    80 16 X 8 QLDPARDVLCL XLXXXXXXXXL Chisari 944
    4.073
    90 18 NUC 99 QLLWFHISCL XLXXXXXXXL 1.0908 * 945
    95 19 POL 685 QVFADATPTGW XVXXXXXXXXW 946
    95 19 ENV 344 QWFVGLSPTVW XWXXXXXXXX 947
    75 15 ENV 242 RFIIFLFI XFXXXXXI 17.0151 948
    75 15 ENV 242 RFIIFLFIL XFXXXXXXL 949
    75 15 ENV 242 RFIIFLFILL XFXXXXXXXL 950
    75 15 ENV 242 RFIIFLFILLL XFXXXXXXXXL 951
    100 20 ENV 332 RFSWLSLL XFXXXXXL 952
    100 20 ENV 332 RFSWLSLLVPF XFXXXXXXXXF 953
    80 16 ENV 187 RILTIPQSL XIXXXXXXL 1.0149 954
    90 18 POL 624 RIVGLLGF XIXXXXXF 955
    75 15 POL 106 RLKLIMPARF XLXXXXXXXF 956
    75 15 POL 106 RLKLIMPARFY XLXXXXXXXXY 957
    95 19 POL 376 RLVVDFSQF XLXXXXXXF 20.0122 958
    90 18 POL 353 RTPARVTGGVF XTXXXXXXXXF 959
    95 19 POL 36 RVAEDLNL XVXXXXXL 960
    90 18 POL 36 RVAEDLNLGNL XVXXXXXXXXL 961
    80 16 POL 818 RVHFASPL XVXXXXXL 962
    100 20 POL 357 RVTGGVFL XVXXXXXL 963
    85 17 POL 577 RWGYSLNF XWXXXXXF 964
    85 17 POL 577 RWGYSLNFM XWXXXXXXM 265
    85 17 POL 577 RWGYSLNFMGY XWXXXXXXXXY 966
    95 19 ENV 236 RWMCLRRF XWXXXXXF 967
    95 19 ENV 236 RWMCLRRFI XWXXXXXXI 20.0138 * 0.0710 968
    95 19 ENV 236 RWMCLRRFII XWXXXXXXXI 20.0269 * 1.1000 969
    95 19 ENV 236 RWMCLRRFIIF XWXXXXXXXXF 970
    100 20 POL 167 SFCGSPYSW XFXXXXXXW 20.0139 * 0.0710 971
    95 19 NUC 46 SFLPSDFF XFXXXXXF 972
    80 16 POL 765 SFVYVPSAL XFXXXXXXL 973
    100 20 POL 49 SIPWTHKVGNF XIXXXXXXXXF 974
    95 19 ENV 194 SLDSWWTSL XLXXXXXXL 1.0150 975
    95 19 ENV 194 SLDSWWTSLNF XLXXXXXXXXF 976
    95 19 POL 416 SLDVSAAF XLXXXXXF 977
    95 19 POL 416 SLDVSAAFY XLXXXXXXY 1.0186 * 978
    100 20 ENV 337 SLLVPFVQW XLXXXXXXW 979
    100 20 ENV 337 SLLVPFVQWF XLXXXXXXXF 980
    75 15 POL 581 SLNFMGYVI XLXXXXXXI 3.0011 981
    95 19 X 54 SLRGLPVCAF XLXXXXXXXF 20.0259 982
    90 18 POL 403 SLTNLLSSNL XLXXXXXXXL 1.0548 983
    75 15 X 104 STTDLEAY XTXXXXXY 984
    75 15 X 104 STTDLEAYF XTXXXXXXF 985
    75 15 ENV 17 SVPNPLGF SVXXXXXF 986
    85 17 POL 548 SVQHLESL XVXXXXXL 987
    80 16 ENV 330 SVRFSWLSL XVXXXXXXL 1.0153 988
    80 16 ENV 330 SVRFSWLSLL XVXXXXXXXL 1.0517 989
    90 18 POL 739 SVVLSRKY XVXXXXXY 26.0029 990
    85 17 POL 739 SVVLSRKYTSF XVXXXXXXXXF 991
    95 19 POL 524 SVVRRAFPHCL XVXXXXXXXXL 992
    95 19 POL 413 SWLSLDVSAAF XWXXXXXXXXF 993
    100 20 ENV 334 SWLSLLVPF XWXXXXXXF 20.0136 * 0.3900 994
    95 19 POL 392 SWPKFAVPNL XWXXXXXXXL 20.0271 * 5.6000 995
    100 20 ENV 197 SWWTSLNF XWXXXXXF 996
    95 19 ENV 197 SWWTSLNFL XWXXXXXXL 20.0137 * 0.3800 997
    90 18 POL 537 SYMDDVVL XYXXXXXL 998
    75 15 POL 4 SYCHFRKL XYXXXXXL 999
    75 15 POL 4 SYCHFRKLL XYXXXXXXL 2.0042 0.0051 1000
    75 15 POL 4 SYCHFRKLLL XYXXXXXXXL 2.0173 * 0.0660 1001
    75 15 POL 4 SYCHFRKLLLL XYXXXXXXXXL 1002
    75 15 NUC 138 TFGRETVL XFXXXXXL 1003
    75 15 NUC 138 TFGRETVLEY XFXXXXXXXY 1004
    75 15 NUC 138 TFGRETVLEYL XFXXXXXXXXL 1005
    95 19 POL 657 TFSPTYKAF XFXXXXXXF 5.0064 0.0060 1006
    95 19 POL 657 TFSPTYKAFL XFXXXXXXXL 5.0117 0.0043 1007
    90 18 ENV 190 TIPQSLDSW XIXXXXXXW 1008
    90 18 ENV 190 TIPQSLDSWW XIXXXXXXXW 1009
    100 20 POL 150 TLWKAGIL XLXXXXXL 1010
    100 20 POL 150 TLWKAGILY XLXXXXXXY 1.0177 * 1011
    75 15 X 105 TTDLEAYF XTXXXXXF 1012
    85 17 POL 798 TTGRTSLY XTXXXXXY 26.0030 1013
    85 17 POL 100 TVNEKRRL XVXXXXXL 1014
    80 16 NUC 16 TVQASKLCL XVXXXXXXL 1.0365 1015
    80 16 NUC 16 TVQASKLCLGW XVXXXXXXXXW 1016
    75 15 ENV 352 TVWLSVIW XVXXXXXW 1017
    75 15 ENV 352 TVWLSVIWM XVXXXXXXM 3.0035 1018
    95 19 POL 686 VFADATPTGW XFXXXXXXXW 20.0272 * 0.0180 1019
    75 15 X 131 VFVLGGCRHKL XFXXXXXXXXL 1020
    85 17 POL 543 VLGAKSVQHL XLXXXXXXXL 1.0560 1021
    90 18 X 133 VLGGCRKHL XLXXXXXXL 1.0220 1022
    85 17 X 92 VLHKRTLGL XLXXXXXXL 1.0391 1023
    95 19 ENV 259 VLLDYQGM XLXXXXXM 17.0107 1024
    95 19 ENV 259 VLLDYQGML XLXXXXXXL 1.0151 * 1025
    95 19 ENV 177 VLQAGFFL XLXXXXXL Chisari 1026
    4.027
    95 19 ENV 177 VLQAGFFLL XLXXXXXXL 1.0828 * 1027
    85 17 POL 741 VLSRKYTSF XLXXXXXXF 1028
    85 17 POL 741 VLSRKYTSFPW XLXXXXXXXXW 1029
    80 16 POL 542 VVLGAKSVQHL XVXXXXXXXXL 1030
    85 17 POL 740 VVLSRKYTSF XVXXXXXXXF 20.9261 1031
    95 19 POL 525 VVRRAFPHCL XVXXXXXXXL 1.0558 1032
    95 19 NUC 124 VWIRTPPAY XWXXXXXXY 1033
    75 15 ENV 353 VWLSVIWM XWXXXXXM 1034
    90 18 NUC 102 WFHISCLTF XFXXXXXXF 13.0073 * 0.0300 1035
    95 19 ENV 345 WFVGLSPTVW XFXXXXXXXW 20.0270 * 0.0120 1036
    95 19 ENV 345 WFVGLSPTVWL XFXXXXXXXXL 1037
    80 16 POL 759 WILRGTSF XIXXXXXF 1038
    80 16 POL 759 WILRGTSFVY XIXXXXXXXY 1.0572 1039
    95 19 NUC 125 WIRTPPAY XIXXXXXY 26.0031 1040
    80 16 POL 751 WLLGCAANW XLXXXXXXW 1041
    80 16 POL 751 WLLGCAANWI XLXXXXXXXI Chisari 1042
    4.104
    80 16 POL 751 WLLGCAANWIL XLXXXXXXXXL 1043
    95 19 POL 414 WLSLDVSAAF XLXXXXXXXF 1044
    95 19 POL 414 WLSLDVSAAFY XLXXXXXXXXY 26.0551 1045
    100 20 ENV 335 WLSLLVPF XLXXXXXF 1046
    100 20 ENV 335 WLSLLVPFVQW XLXXXXXXXXW 1047
    85 17 NUC 26 WLWGMDIDPY XLXXXXXXXY 1.0774 * 1048
    95 19 ENV 237 WMCLRRFI XMXXXXXI 1049
    95 19 ENV 237 WMCLRRFII XMXXXXXXI 3.0031 * 0.0230 1050
    95 19 ENV 237 WMCLRRFIIF XMXXXXXXXF 20.0266 0.0013 1051
    95 19 ENV 237 WMCLRRFIIFL XMXXXXXXXXL Chisari 1052
    4.024
    85 17 ENV 359 WMMWYWGPSL XMXXXXXXXL 1.0901 * 0.0005 1053
    85 17 ENV 359 WMMWYWGPSL XMXXXXXXXXY 26.0552 * 1054
    100 20 POL 52 WTHKVGNF XTXXXXXF 1055
    95 19 POL 52 WTHKVGNFTGL XTXXXXXXXXL 1056
    95 19 ENV 198 WWTSLNFL XWXXXXXL 1057
    85 17 ENV 362 WYWGPSLY XYXXXXXY 3.0362 0.0001 1058
    100 20 POL 147 YLHTLWKAGI XLXXXXXXXI 7.0066 * 1059
    100 20 POL 147 YLHTLWKAGIL XLXXXXXXXXL 1060
    100 20 POL 122 YLPLDKGI XLXXXXXI 1061
    100 20 POL 122 YLPLDKGIKPY XLXXXXXXXXY 26.0553 1062
    90 18 NUC 118 YLVSFGVW XLXXXXXW 1063
    90 18 NUC 118 YLVSFGVWI XLXXXXXXL 3.0007 * 1064
    85 17 POL 746 YTSFPWLL XTXXXXXL 1065
    411
  • TABLE X
    HBV B07 SUPER MOTIF (With binding information)
    C-
    Conservancy Frequency Protein Position Sequence P2 term Peptide AA Filed B*0702 B*3501
    75 15 X 146 APCNFFTSA P A 9
    95 19 POL 633 APFTQOCY P Y 19.0013 8 0.0001 0.0012
    95 19 POL 633 APFTQCGYPA P A 16.0180 10 * 0.0029 0.0001
    95 19 POL 633 APFTQCGYPAL P L 26.0554 11 * 0.2300 0.0010
    100 20 ENV 232 CPGYRWMCL P L 1308.21 9
    80 16 NUC 14 CPTVQASKL P L 9
    80 16 NUC 14 CPTVQASKLCL P L 11
    80 16 X 10 DPARDVLCL P L 9
    80 16 ENV 122 DPRVRGLY P Y 8
    90 18 POL 778 DPSRGRLGL P L 1147.01 9 * 0.0120 0.0001
    90 18 NUC 33 DPYKEFGA P A 19.0008 8 0.0001 0.0001
    75 15 ENV 130 FPAGGSSSGTV P V 11
    90 18 ENV 14 FPDHQLDPA P A 1308.23 9 *
    85 17 ENV 14 FPDHQLDPAF P F 20.0274 10 0.0002 0.0016
    95 19 POL 530 FPHCLAFSY P Y 1145.08 9 * 0.0001 0.5250
    95 19 POL 530 FPHCLAFSYM P M 1147.05 10 * 0.0990 0.2200
    75 15 POL 749 FPWLLGCA P A 8
    75 15 POL 749 FPVVLLGCAA P A 9
    75 15 POL 749 FPWLLGCAANW P W 11
    90 18 X 67 GPCALRFTSA P A 16.0182 10 * 0.0900 0.0001
    95 19 POL 19 GPLEEELPRL P L 15.0208 10 0.0001 0.0001
    90 18 POL 19 GPLEEELPRLA P A 26.0555 11 −0.0002 0.0001
    95 19 ENV 173 GPLLVLQA P A 19.0003 8 * 0.0003 0.0001
    95 19 ENV 173 GPLLVLQAGF P F 15.0212 10 0.0001 0.0001
    95 19 ENV 173 GPLLVLQAGFF P F 26.0556 11 0.0011 0.0001
    85 17 POL 97 GPLTVNEKRRL P L 26.0557 11 0.0031 0.0001
    100 20 POL 429 HPAAMPHL P L 19.0011 8 * 0.0650 0.0004
    100 20 POL 429 HPAAMPHLL P L 1147.02 9 * 0.0980 0.0270
    85 17 POL 429 HPAAMPHLLV P V 20.0273 10 * 0.0160 0.0020
    80 16 POL 495 HPIILGFRKI P I 10
    100 20 ENV 313 IPIPSSWA P A 19.0005 8 * 0.0004 0.0004
    100 20 ENV 313 IPIPSSWAF P F 1145.04 9 * 0.1300 2.7679
    80 16 ENV 313 IPIPSSWAFA P A 16.0177 10 * 0.0013 0.0024
    80 16 POL 504 IPMGVGLSPF P F 10
    80 16 POL 504 IPMGVGLSPFL P L 11
    90 18 ENV 191 IPQSLDSW P W F126.65 8
    90 18 ENV 191 IPQSLDSWW P W F126.60 9 *
    80 16 ENV 315 IPSSWAFA P A 8
    100 20 POL 50 IPWTHKVGNF P F 15.0209 10 0.0013 0.0001
    100 20 ENV 379 LPIFFCLW P W 19.0007 8 * 0.0001 0.0001
    100 20 ENV 379 LPIFFCLWV P V 1308.22 9 *
    100 20 ENV 379 LPIFFCLWVY P Y 15.0215 10 0.0002 0.0079
    100 20 ENV 379 LPIFFCLWVYI P I 26.0558 11 0.0002 0.0001
    85 17 POL 712 LPIHTAEL P L 17.0259 8
    85 17 POL 712 LPIHTAELL P L 20.0140 9 * 0.0040 0.0630
    85 17 POL 712 LPIHTAELLA P A 16.0181 10 * 0.0018 0.0011
    85 17 POL 712 LPIHTAELLAA P A 26.0559 11 0.0090 0.0027
    80 16 X 89 LPKVLHKRTL P L 10
    100 20 POL 123 LPLDKGIKPY P Y 15.0210 10 * 0.0001 0.0290
    100 20 POL 123 LPLDKGIKPYY P Y 26.0560 11 −0.0002 0.0009
    95 19 X 58 LPVCAFSSA P A 1147.06 9 * 0.0480 0.0710
    80 16 POL 611 LPVNRPIDW P W 9
    80 16 POL 611 LPVNRPIDWKV P V 11
    80 16 POL 433 MPHLLVGSSGL P L 11
    100 20 POL 1 MPLSYQHF P F 19.0010 8 * 0.0001 0.0097
    75 15 POL 1 MPLSYQHFRKL P L 11
    90 18 POL 774 NPADDPSRGRL P L 26.0561 11 * 0.0120 0.0001
    95 19 ENV 9 NPLGFFPDHQL P L 26.0562 11 0.0012 0.0021
    75 15 POL 571 NPNKTKRW P W 8
    75 15 POL 571 NPNKTKRWGY P Y 10
    95 19 NUC 129 PPAYRPPNA P A 16.0007 9 0.0001 0.0001
    95 19 NUC 129 PPAYRPPNAPI P I 26.0563 11
    85 17 ENV 58 PPHGGLLGW P W 20.0141 9 0.0001 0.0002
    100 20 NUC 134 PPNAPILSTL P L 15.0211 10 0.0001 0.0001
    80 16 POL 615 RPIDWKVCQRI P I 11
    100 20 NUC 133 RPPNAPIL P L 19.0009 8 * 0.0078 0.0001
    100 20 NUC 133 RPPNAPILSTL P L 26.0564 11 * 0.1300 0.0001
    100 20 NUC 44 SPEHCSPHHTA P A 26.0565 11 −0.0002 0.0001
    95 19 POL 511 SPFLLAQF P F 19.0012 8 * 0.5500 0.0009
    95 19 POL 511 SPFLLAQFTSA P A 26.0566 11 * 0.0820 0.0001
    100 20 NUC 49 SPHHTALRQA P A 16.0178 10 0.0012 0.0001
    100 20 NUC 49 SPHHTALRQAI P I 26.0567 11 * 0.5800 0.0001
    85 17 ENV 67 SPQAQGIL P L 8
    85 17 POL 808 SPSVPSHL P L 8
    75 15 ENV 350 SPTVWLSV P V 8
    75 15 ENV 350 SPTVWLSVI P I 1308.16 9
    75 15 ENV 350 SPTVWLSVIW P W 1308.17 10
    75 15 ENV 350 SPTVWLSVIWM P M 11
    95 19 POL 659 SPTYKAFL P L 19.0015 8 * 0.3900 0.0001
    90 18 POL 354 TPARVTGGV P V 1147.07 9 * 0.0078 0.0001
    90 18 POL 354 TPARVTGGVF P F 1147.04 10 * 0.3200 0.1000
    90 18 POL 354 TPARVTGGVFL P L 26.0568 11 * 0.0950 0.0001
    95 19 NUC 128 TPPAYRPPNA P A 16.0179 10 * 0.0001 0.0001
    75 15 ENV 57 TPPHGGLL P L 8
    75 15 ENV 57 TPPHGGLLGW P W 1308.04 10
    80 16 POL 691 TPTGWGLA P A 8
    75 15 POL 691 TPTGWGLAI P I 9
    95 19 ENV 340 VPFVQWFV P V 19.0006 8 * 0.0010 0.0001
    95 19 ENV 340 VPFVQNFVGL P L 15.0213 10 0.0011 0.0001
    95 19 POL 398 VPNLQSLTNL P L 15.0216 10 0.0006 0.0001
    95 19 POL 396 VPNLQSLTNLL P L 26.0569 11 0.0004 0.0001
    90 18 POL 769 VPSALNPA P A 19.0016 8 * 0.0011 0.0001
    95 19 POL 393 WPKFAVPNL P L 15.0035 9 0.0054 0.0002
    95 19 POL 640 YPALMPLY P Y 19.0014 8 * 0.0004 0.2600
    95 19 POL 640 YPALMPLYA P A 1147.08 9 * 0.0180 0.0480
    95 19 POL 640 YPALMPLYACI R I 26.0570 11 0.0040 0.0001
    SEQ
    ID
    Conservancy Frequency Protein Position Sequence B*5101 B*5301 B*5401 NO:
    75 15 X 146 APCNFFTSA 1066
    95 19 POL 633 APFTQOCY 0.0019 0.0002 0.0002 1067
    95 19 POL 633 APFTQCGYPA 0.0002 1.4000 1068
    95 19 POL 633 APFTQCGYPAL 0.0004 −0.0003 0.0093 1069
    100 20 ENV 232 CPGYRWMCL 1070
    80 16 NUC 14 CPTVQASKL 1071
    80 16 NUC 14 CPTVQASKLCL 1072
    80 16 X 10 DPARDVLCL 1073
    80 16 ENV 122 DPRVRGLY 1074
    90 18 POL 778 DPSRGRLGL 0.0001 0.0001 0.0001 1075
    90 18 NUC 33 DPYKEFGA 0.0019 0.0002 0.0019 1076
    75 15 ENV 130 FPAGGSSSGTV 1077
    90 18 ENV 14 FPDHQLDPA 1078
    85 17 ENV 14 FPDHQLDPAF 0.0003 0.0011 0.0021 1079
    95 19 POL 530 FPHCLAFSY 0.0665 0.5400 0.0199 1080
    95 19 POL 530 FPHCLAFSYM 0.0900 0.0790 0.0480 1081
    75 15 POL 749 FPWLLGCA 1082
    75 15 POL 749 FPVVLLGCAA 1083
    75 15 POL 749 FPWLLGCAANW 1084
    90 18 X 67 GPCALRFTSA 0.0001 0.0002 0.0035 1085
    95 19 POL 19 GPLEEELPRL 0.0002 0.0001 0.0002 1086
    90 18 POL 19 GPLEEELPRLA 0.0001 −0.0003 0.0001 1087
    95 19 ENV 173 GPLLVLQA 0.0110 0.0002 0.0065 1088
    95 19 ENV 173 GPLLVLQAGF 0.0002 0.0001 0.0002 1089
    95 19 ENV 173 GPLLVLQAGFF 0.0001 0.0008 0.0009 1090
    85 17 POL 97 GPLTVNEKRRL 0.0001 −0.0003 0.0001 1091
    100 20 POL 429 HPAAMPHL 0.3100 0.0037 0.0160 1092
    100 20 POL 429 HPAAMPHLL 0.0110 0.0500 0.0120 1093
    85 17 POL 429 HPAAMPHLLV 0.0078 0.0140 0.0170 1094
    80 16 POL 495 HPIILGFRKI 1095
    100 20 ENV 313 IPIPSSWA 0.0019 0.0002 0.0600 1096
    100 20 ENV 313 IPIPSSWAF 2.3500 0.7450 0.0034 1097
    80 16 ENV 313 IPIPSSWAFA 0.0014 0.4500 1098
    80 16 POL 504 IPMGVGLSPF 1099
    80 16 POL 504 IPMGVGLSPFL 1100
    90 18 ENV 191 IPQSLDSW 1101
    90 18 ENV 191 IPQSLDSWW 1102
    80 16 ENV 315 IPSSWAFA 1103
    100 20 POL 50 IPWTHKVGNF 0.0007 0.0001 0.0002 1104
    100 20 ENV 379 LPIFFCLW 0.0360 0.1400 0.0035 1105
    100 20 ENV 379 LPIFFCLWV 1106
    100 20 ENV 379 LPIFFCLWVY 0.0002 0.0006 0.0002 1107
    100 20 ENV 379 LPIFFCLWVYI 0.0043 0.0139 0.0021 1108
    85 17 POL 712 LPIHTAEL 1109
    85 17 POL 712 LPIHTAELL 0.0052 0.3100 0.0005 1110
    85 17 POL 712 LPIHTAELLA 0.0016 0.3300 1111
    85 17 POL 712 LPIHTAELLAA −0.0003 0.0120 2.7500 1112
    80 16 X 89 LPKVLHKRTL 1113
    100 20 POL 123 LPLDKGIKPY 0.0002 0.0003 0.0002 1114
    100 20 POL 123 LPLDKGIKPYY 0.0001 0.0007 0.0001 1115
    95 19 X 58 LPVCAFSSA 0.0110 0.0009 19.0000 1116
    80 16 POL 611 LPVNRPIDW 1117
    80 16 POL 611 LPVNRPIDWKV 1118
    80 16 POL 433 MPHLLVGSSGL 1119
    100 20 POL 1 MPLSYQHF 0.0120 0.0370 0.0190 1120
    75 15 POL 1 MPLSYQHFRKL 1121
    90 18 POL 774 NPADDPSRGRL 0.0001 −0.0003 0.0001 1122
    95 19 ENV 9 NPLGFFPDHQL 0.0001 0.0028 0.0001 1123
    75 15 POL 571 NPNKTKRW 1124
    75 15 POL 571 NPNKTKRWGY 1125
    95 19 NUC 129 PPAYRPPNA 0.0001 0.0002 0.0003 1126
    95 19 NUC 129 PPAYRPPNAPI 0.0001 −0.0003 0.0001 1127
    85 17 ENV 58 PPHGGLLGW 0.0001 0.0003 0.0002 1128
    100 20 NUC 134 PPNAPILSTL 0.0035 0.0001 0.0002 1129
    80 16 POL 615 RPIDWKVCQRI 1130
    100 20 NUC 133 RPPNAPIL 0.0280 0.0002 0.0002 1131
    100 20 NUC 133 RPPNAPILSTL 0.0018 −0.0003 0.0001 1132
    100 20 NUC 44 SPEHCSPHHTA 0.0001 −0.0003 0.0011 1133
    95 19 POL 511 SPFLLAQF 0.0180 0.0009 0.0093 1134
    95 19 POL 511 SPFLLAQFTSA 0.0001 −0.0003 12.0500 1135
    100 20 NUC 49 SPHHTALRQA 0.0002 0.0035 1136
    100 20 NUC 49 SPHHTALRQAI 0.0004 0.0005 0.0002 1137
    85 17 ENV 67 SPQAQGIL 1138
    85 17 POL 808 SPSVPSHL 1139
    75 15 ENV 350 SPTVWLSV 1140
    75 15 ENV 350 SPTVWLSVI 1141
    75 15 ENV 350 SPTVWLSVIW 1142
    75 15 ENV 350 SPTVWLSVIWM 1143
    95 19 POL 659 SPTYKAFL 0.0019 0.0002 0.0002 1144
    90 18 POL 354 TPARVTGGV 0.0013 0.0001 0.0015 1145
    90 18 POL 354 TPARVTGGVF 0.0001 0.0099 0.0006 1146
    90 18 POL 354 TPARVTGGVFL 0.0001 0.0005 0.0005 1147
    95 19 NUC 128 TPPAYRPPNA 0.0002 0.0100 1148
    75 15 ENV 57 TPPHGGLL 1149
    75 15 ENV 57 TPPHGGLLGW 1150
    80 16 POL 691 TPTGWGLA 1151
    75 15 POL 691 TPTGWGLAI 1152
    95 19 ENV 340 VPFVQWFV 19.0000 0.0002 0.1100 1153
    95 19 ENV 340 VPFVQNFVGL 0.0100 0.0001 0.0025 1154
    95 19 POL 398 VPNLQSLTNL 0.0004 0.0001 0.0002 1155
    95 19 POL 396 VPNLQSLTNLL 0.0001 −0.0003 0.0002 1156
    90 18 POL 769 VPSALNPA 0.0070 0.0002 1.0000 1157
    95 19 POL 393 WPKFAVPNL 0.0015 0.0001 0.0015 1158
    95 19 POL 640 YPALMPLY 0.4100 0.0450 0.0056 1159
    95 19 POL 640 YPALMPLYA 0.0340 0.0140 16.0000 1160
    95 19 POL 640 YPALMPLYACI 0.0470 0.0320 0.0700 1161
    96
  • TABLE XI
    HBV B27 SUPER MOTIF
    SEQ ID
    Source Conservancy Freq Protein Position Sequence String Supermotif Peptide Filed NO:
    HBV 95 19 X 51 AHLSLRGL XHXXXXXL B27s 1162
    HBV 85 17 POL 546 AKSVCHLESL XKXXXXXXXL B27s 1163
    HBV 90 18 POL 356 ARVTGGVF XRXXXXXF B27s 1164
    HBV 90 18 POL 356 ARVTGGVFL XXXXXXXL B27s 1165
    HBV 95 19 X 48 DHGAHLSL XHXXXXXL B27s 1166
    HBV 95 19 X 48 DHGAHLSLRGL XHXXXXXXXXL B27s 1167
    HBV 90 18 ENV 16 DHQLDPAF XHXXXXXF B27s 1168
    HBV 100 20 POL 126 DKGIKPYY XKXXXXXY B27s 1169
    HBV 100 20 NUC 46 EHCSPHHTAL XHXXXXXXXL B27s 1170
    HBV 90 16 NUC 103 FHISCLTF XHXXXXXF B27s 1171
    HBV 80 16 POL 501 FRKIPMGVGL XRXXXXXXXL B27s 1172
    HBV 80 16 POL 608 FRKLOVNRPI XRXXXXXXXI B27s 1173
    HBV 75 15 NUC 140 GRETVLEY XRXXXXXY B27s 1174
    HBV 75 15 NUC 140 GRETVLEYL XRXXXXXXL B27s 1175
    HBV 100 20 NUC 51 HHTALRQAI XHXXXXXXI B27s 1176
    HBV 95 19 NUC 51 HHTALRQAIL XHXXXXXXXL B27s 1177
    HBV 95 19 POL 54 HKVGNFTGL XKXXXXXXL B27s 17.0358 1178
    HBV 95 19 POL 54 HKVGNFTGLY XKXXXXXXXY B27s 1179
    HBV 75 15 POL 568 IHLNPNKTKRW XHXXXXXXXXW B27s 1180
    HBV 85 17 POL 714 IHTAELLAACF XHXXXXXXXXF B27s 1181
    HBV 85 17 POL 576 KRWGYSLNF XRXXXXXXF B27s 1182
    HBV 85 17 POL 576 KRWGYSLNFM XRXXXXXXXM B27s 1183
    HBV 90 18 X 93 LHKRTLGL XHXXXXXL B27s 1184
    HBV 95 19 POL 490 LHLYSHPI XHXXXXXI B27s 1185
    HBV 80 16 POL 490 LHLYSHPII XHXXXXXXI B27s 1186
    HBV 80 16 POL 490 LHLYSHPIIL XHXXXXXXXL B27s 1187
    HBV 100 20 POL 428 LHPAAMPHL XHXXXXXXL B27s 1188
    HBV 100 20 POL 428 LHPAAMPHLL XHXXXXXXXL B27s 1189
    HBV 100 20 POL 148 LHTLWKAGI XHXXXXXXI B27s 1190
    HBV 100 20 POL 148 LHTLWKAGIL XHXXXXXXXL B27s 1191
    HBV 100 20 POL 148 LHTLWKAGILY XHXXXXXXXXY B27s 1192
    HBV 75 15 POL 107 LKLIMPARF XKXXXXXXF B27s 1193
    HBV 75 15 POL 107 LKLIMPARFY XKXXXXXXXY B27s 1194
    HBV 95 19 POL 55 LRGLPVCAF XRXXXXXXF B27s 1195
    HBV 80 16 NUC 761 LRGTSFVY XRXXXXXY B27s 1196
    HBV 95 19 NUC 55 LRQAILCW XRXXXXXW B27s 1197
    HBV 90 18 ENV 55 LRQAILCWGEL XRXXXXXXXXL B27s 1198
    HBV 95 19 ENV 240 LRRFIIFL XRXXXXXL B27s 1199
    HBV 75 15 ENV 240 LRRFIIFLF XRXXXXXXF B27s 1200
    HBV 75 15 ENV 240 LRRFIIFLFI XRXXXXXXXI B27s 1201
    HBV 75 15 POL 240 LRRFIIFLFIL XRXXXXXXXXL B27s 1202
    HBV 75 15 POL 573 NKTKRWGY XKXXXXXY B27s 1203
    HBV 75 15 POL 573 NKTKRWGYSL XKXXXXXXXL B27s 1204
    HBV 85 17 POL 34 NRRVAEDL XRXXXXXL B27s 1206
    HBV 85 17 POL 34 NRRVAEDLNL XRXXXXXXXL B27s 1206
    HBV 95 19 POL 531 PHCLAFSY XHXXXXXY B27s 1207
    HBV 95 19 POL 531 PHCLAFSYM XHXXXXXXM B27s 1208
    HBV 85 17 ENV 59 PHGGLLGW XHXXXXXW B27s 1209
    HBV 100 20 NUC 50 PHHTALRQAI XHXXXXXXXI B27s 1210
    HBV 95 19 NUC 50 PHHTALRQAIL XHXXXXXXXXL B27s 1211
    HBV 80 16 POL 434 PHLLVGSSGL XHXXXXXXXL B27s 1212
    HBV 95 19 POL 394 PKFAVPNL XKXXXXX B27s 1213
    HBV 95 19 POL 394 PKFAVPNLQSL XKXXXXXXXXL B27s 1214
    HBV 85 17 X 90 PKVLHKRTL XKXXXXXXL B27s 1215
    HBV 85 17 X 90 PKVLHKRTLGL XKXXXXXXXXL B27s 1216
    HBV 75 15 POL 6 QHFRKLLL XHXXXXXL B27s 1217
    HBV 75 15 POL 6 QHFRKLLLL XHXXXXXXL B27s 1218
    HBV 90 18 POL 623 QRIVGLLGF XRXXXXXXF B27s 1219
    HBV 100 20 POL 145 RHYLHTLW XHXXXXXW B27s 1220
    HBV 80 16 POL 502 RKIPMGVGL XKXXXXXXL B27s 1221
    HBV 80 16 POL 609 RKLPVNRPI XKXXXXXXI B27s 1222
    HBV 80 16 POL 609 RKLPVNRPIDW XKXXXXXXXXW B27s 1223
    HBV 85 17 POL 744 RKYTSFPW XKXXXXXW B27s 1224
    HBV 85 17 POL 744 RKYTSFPWL XKXXXXXXL B27s 1225
    HBV 85 17 POL 744 RKYTSFPWLL XKXXXXXXXL B27s 1226
    HBV 95 19 POL 527 RRAFPHCL XRXXXXXL B27s 1227
    HBV 95 19 POL 527 RRAFPHCLAF XRXXXXXXXF B27s 1228
    HBV 75 15 ENV 241 RRFIIFLF XRXXXXXF B27s 1229
    HBV 75 15 ENV 241 RRFIIFLFI XRXXXXXXI B27s 1230
    HBV 75 15 ENV 241 RRFIIFLFIL XRXXXXXXXL B27s 1231
    HBV 75 15 ENV 241 RRFIIFLFILL XRXXXXXXXXL B27s 1232
    HBV 75 15 POL 105 RRLKLIMPARF XRXXXXXXXXF B27s 1233
    HBV 90 18 POL 35 RRVAEDLNL XRXXXXXXL B27s 1234
    HBV 80 16 POL 494 SHPIILGF XHXXXXXF B27s 1235
    HBV 80 16 POL 494 SHPIILGFRKI XHXXXXXXXXI B27s 1236
    HBV 90 18 NUC 20 SKLCLGWL XKXXXXXL B27s 1237
    HBV 85 17 NUC 20 SKLCLGWLW XKXXXXXXW B27s 1238
    HBV 85 17 NUC 20 SKLCLGWLWGM XKXXXXXXXXM B27s 1239
    HBV 85 17 POL 743 SRKYTSFPW XRXXXXXXW B27s 1240
    HBV 85 17 POL 743 SRKYTSFPWL XRXXXXXXXL B27s 1241
    HBV 85 17 POL 743 SPKYTSFPWLL XRXXXXXXXXL B27s 1242
    HBV 95 19 POL 375 SPLVVDFSQF XRXXXXXXXF B27s 1243
    HBV 80 16 POL 472 SRNLYVSL XRXXXXXL B27s 17.0123 1244
    HBV 95 19 POL 53 THKVGNFTGL XHXXXXXXXL B27s 1245
    HBV 95 19 POL 53 THKVGNFTGLY XHXXXXXXXXY B27s 1246
    HBV 95 19 POL 575 TKRWGYSL XKXXXXXL B27s 1247
    HBV 85 17 POL 575 TKRWGYSLNF XKXXXXXXXF B27s 1248
    HBV 85 17 POL 575 TKRWGYSLNFM XKXXXXXXXXM B27s 1249
    HBV 100 20 POL 120 TKYLPLDKGI XKXXXXXXXI B27s 1250
    HBV 100 20 POL 144 TRHYLHTL XRXXXXXL B27s 1251
    HBV 100 20 POL 144 TRHYLHTLW XRXXXXXXW B27s 1252
    HBV 80 16 ENV 186 TRILTIPQSL XRXXXXXXXL B27s 1253
    HBV 80 16 POL 819 VHFASPHVAW XHXXXXXXXXW B27s 1254
    HBV 80 16 ENV 331 VRFSWLSL XRXXXXXL B27s 1255
    HBV 80 16 ENV 331 VRFSWLSLL XRXXXXXXL B27s 1256
    HBV 95 19 POL 526 VRRAFPHCL XRXXXXXXL B27s 1257
    HBV 95 19 POL 526 VRRAFPHCLAF XRXXXXXXXXF B27s 1258
    HBV 85 17 POL 619 WKVCQRIVGL XKXXXXXXXL B27s 1259
    HBV 85 17 POL 619 WKVCQRIVGLL XKXXXXXXXXL B27s 1260
    HBV 100 20 NUC 132 YRPPNAPI XRXXXXX B27s 1261
    HBV 100 20 NUC 132 YRPPNAPIL XRXXXXXX B27s 17.0356 1262
    HBV 95 19 ENV 235 YRWMCLRRF XRXXXXXXF B27s 1263
    HBV 95 19 ENV 235 YRWMCLRRFI XRXXXXXXXI B27s 1264
    HBV 95 19 ENV 235 YRWMCLRRFII XRXXXXXXXXI B27s 1265
    104
  • TABLE XII
    HBV B44 SUPER MOTIF
    SEQ ID
    Source Conservancy Freq Protein Position Sequence String Supermotif Peptide Filed NO:
    HBV 95 19 POL 688 ADATPTGW XDXXXXXW B44 1266
    HBV 95 19 POL 688 ADATPTGWGL XDXXXXXXXL B44 1267
    HBV 80 16 POL 688 ADATPTGWGL XDXXXXXXXXA B44 1268
    HBV 90 18 POL 776 ADDPSRGRL XDXXXXXXL B44 1269
    HBV 90 18 POL 776 ADDPSRGRLGL XDXXXXXXXXL B44 1270
    HBV 95 19 POL 38 AEDUNLGNL XEXXXXXXL B44 17.0357 1271
    HBV 95 19 POL 38 AEDUNLGNUNV XEXXXXXXXXV B44 1272
    HBV 85 17 POL 717 AELLAACF XEXXXXXF B44 1273
    HBV 85 17 POL 717 AELLAACFA XEXXXXXXA B44 1274
    HBV 90 18 POL 777 DDPSRGRL XDXXXXXL B44 17.0010 1278
    HBV 90 18 POL 777 DDPSRGRGL XDXXXXXXXL B44 17.0418 1276
    HBV 90 18 POL 540 DDVVLGAKSV XDXXXXXXXV B44 1277
    HBV 75 15 POL 16 DEAGPLEEEL XEXXXXXXXL B44 1278
    HBV 95 19 POL 39 EDUNLGNL XDXXXXXL B44 1279
    HBV 95 19 POL 39 EDUNLGNLNV XDXXXXXXXV B44 1280
    HBV 90 18 POL 22 EEELPRLA XEXXXXXA B44 1281
    HBV 80 16 X 121 EELGEEIPL XEXXXXXXL B44 1282
    HBV 90 18 NUC 32 IDPYKEFGA XDXXXXXXA B44 1283
    HBV 85 17 POL 617 IDWKVOQRI XDXXXXXXI B44 1284
    HBV 85 17 POL 617 IDWKVOQRIV XDXXXXXXXV B44 1285
    HBV 100 20 POL 125 LDKGIKPY XDXXXXXY B44 1286
    HBV 100 20 POL 125 LDKGIKPYY XDXXXXXXY B44 1287
    HBV 80 16 X 9 LDPARDVL XDXXXXXL B44 17.0012 1288
    HBV 80 16 X 9 LDPARDVLCL XDXXXXXXXL B44 17.0419 1289
    HBV 95 19 ENV 195 LDSWWTSL XDXXXXXL B44 1290
    HBV 95 19 ENV 195 LDSWWTSLNF XDXXXXXXXF B44 1291
    HBV 90 18 ENV 195 LDSWWTSLNFL XDXXXXXXXXL B44 1292
    HBV 85 17 NUC 31 LDTASALY XDXXXXXY B44 1293
    HBV 80 16 NUC 31 LDTASALYREA XDXXXXXXXXA B44 1294
    HBV 95 19 POL 417 LDVSAAFY XDXXXXXY B44 1295
    HBV 90 18 ENV 261 LDYQGMLPV XDXXXXXXV B44 1296
    HBV 95 19 POL 21 LEEELPRL XEXXXXXL B44 1297
    HBV 90 18 POL 21 LEEELPRLA XEXXXXXXA B44 1298
    HBV 90 18 POL 539 MDDVVLGA XDXXXXXA B44 1299
    HBV 90 18 POL 539 MDDVVLGAKSV XDXXXXXXXXV B44 1300
    HBV 90 18 NUC 30 MDIDPYKEF XDXXXXXXF B44 1301
    HBV 90 18 NUC 30 MDIDPYKEFGA XDXXXXXXXXA B44 1302
    HBV 95 19 ENV 15 PDHQLDPA XDXXXXXA B44 1303
    HBV 80 18 ENV 15 PDHQLDPAF XDXXXXXXF B44 1304
    HBV 100 20 NUC 45 PEHCSPHHTA XEXXXXXXXA B44 1305
    HBV 100 20 NUC 45 PEHCSPHHTAL XEXXXXXXXXL B44 1306
    HBV 85 17 NUC 28 RDLLDTASA XDXXXXXXA B44 1307
    HBV 85 17 NUC 28 RDLLDTASAL XDXXXXXXXL B44 1308
    HBV 85 17 NUC 28 RDLLDTASALY XDXXXXXXXXY B44 1309
    HBV 95 19 X 13 RDVLCLRPV XDXXXXXXV B44 1310
    HBV 95 19 X 13 RDVLCLRPVGA XDXXXXXXXXA B44 1311
    HBV 75 15 NUC 141 RETVLEYL XEXXXXXL B44 1312
    HBV 75 15 NUC 141 RETVLEYLV XEXXXXXXV B44 1313
    HBV 90 18 POL 736 TDNSVVLSRKY XDXXXXXXXXY B44 1314
    HBV 95 19 NUC 42 VELLSFLPSDF XEXXXXXXXXF B44 1315
    HBV 80 16 X 120 WEELGEB XEXXXXXI B44 1316
    HBV 80 16 X 120 WEELGEERL XEXXXXXXXL B44 1317
    52
  • TABLE XIII
    HBV B58 SUPER MOTIF
    SEQ ID
    Source Conservancy Freq Protein Position Sequence String Supermotif Peptide Filed NO:
    HBV 85 17 POL 431 AAMPHLLV XAXXXXXV B58 1318
    HBV 95 19 POL 632 AAPFTQCGY XAXXXXXXV B58 1319
    HBV 85 17 NUC 34 ASALYREAL XSXXXXXXL B58 1320
    HBV 100 20 POL 166 ASFCGSPY XSXXXXXY B58 26.0026 * 1321
    HBV 100 20 POL 166 ASFCGSPYSW XSXXXXXXXW B58 1322
    HBV 90 18 NUC 19 ASKLCLGW XSXXXXXW B58 1323
    HBV 90 18 NUC 19 ASKLCLGWL XSXXXXXXL B58 1324
    HBV 85 17 NUC 19 ASKLCLGWLW XSXXXXXXXW B58 1325
    HBV 80 16 POL 822 ASPLHVAW XSXXXXXW B58 1326
    HBV 80 16 ENV 329 ASVRFSWL XSXXXXXL B58 1327
    HBV 80 16 ENV 329 ASVRFSWLSL XSXXXXXXXL B58 1328
    HBV 80 16 ENV 329 ASVRFSWLSLL XSXXXXXXXXL B58 1329
    HBV 95 19 POL 690 ATPTGWGL XTXXXXXL B58 1330
    HBV 75 15 POL 690 ATPTGWGLAI XTXXXXXXXI B58 1331
    HBV 95 19 X 61 CAFSSAGPCAL XSXXXXXL B58 1332
    HBV 100 20 NUC 48 CSPHHTAL XSXXXXXL B58 1333
    HBV 80 16 POL 471 CSRNLYVSL XSXXXXXXL B58 1334
    HBV 95 19 POL 523 CSVVRRAF XSXXXXXF B58 1335
    HBV 100 20 ENV 310 CTCIPIPSSW XTXXXXXXXW B58 1336
    HBV 95 19 POL 689 DATPTGWDL XAXXXXXXL B58 5.0027 1337
    HBV 75 15 POL 689 DATPTGWGLAI XAXXXXXXXXI B58 1338
    HBV 95 19 ENV 196 DSWWTSLNF XSXXXXXXF B58 20.0120 1339
    HBV 90 18 ENV 196 DSWWTSLNFL XSXXXXXXXL B58 1340
    HBV 80 16 NUC 32 DTASALYREA XTXXXXXXXA B58 1341
    HBV 80 16 PUC 32 DTASALYREAL XTXXXXXXXXL B58 1342
    HBV 100 20 POL 17 EAGPLEEEL XAXXXXXXL B58 5.0028 1343
    HBV 95 19 POL 374 ESRLVVDF XSXXXXXF B58 1344
    HBV 95 19 POL 374 ESRLVVDFSQF XSXXXXXXXXF B58 1345
    HBV 75 15 NUC 142 ETVLEYLV XTXXXXXV B58 1346
    HBV 95 19 POL 631 FAAPFTQCGY XAXXXXXXXY B58 20.0254 * 1347
    HBV 95 19 POL 687 FADATPTGW XAXXXXXXW B58 1348
    HBV 95 19 POL 687 FADATPTGWGL XAXXXXXXXXL B58 1349
    HBV 80 16 POL 821 FASPLHVAW XAXXXXXXW B58 1350
    HBV 95 19 POL 396 FAVPNLQSL XAXXXXXXL B58 5.0029 * 1351
    HBV 95 19 POL 658 FSPTYKAF XSXXXXXF B58 1352
    HBV 95 19 POL 658 FSPTYKAFL XSXXXXXXL B58 1353
    HBV 95 19 X 63 FSSAGPCAL XSXXXXXX B58 1354
    HBV 90 18 X 63 FSSAGPCALRF XSXXXXXXXXF B58 1355
    HBV 100 20 ENV 333 FSWLSLLV XSXXXXXV B58 1356
    HBV 100 20 ENV 333 FSWLSLLVPF XSXXXXXXXF B58 20.0263 1357
    HBV 100 20 ENV 333 FSWLSLLVPFV XSXXXXXXXXV B58 1358
    HBV 90 18 POL 538 FSYMDDVV XSXXXXXV B58 17.0257 1359
    HBV 90 18 POL 536 FSYMDDVVL XSXXXXXXL B58 1360
    HBV 95 19 POL 656 FTFSPTYKAF XTXXXXXXXF B58 20.0262 1361
    HBV 95 19 POL 656 FTFSPTYKAFL XTXXXXXXXXL B58 1362
    HBV 90 18 POL 59 FTGLYSSTV XTXXXXXXV B58 20.0118 1363
    HBV 95 19 POL 635 FTQCGYPAL XTXXXXXXL B58 5.0031 1364
    HBV 190 38 POL 635 FTQCGYPALM XTXXXXXXXM B58 5.0085 1365
    HBV 95 19 POL 518 FTSAICSV XTXXXXXV B58 1366
    HBV 95 19 POL 518 FTSAICSVV XTXXXXXXV B58 5.0032 1367
    HBV 95 19 X 50 GAHLSLRGL XAXXXXXXL B58 5.0040 1368
    HBV 90 18 X 50 GAHLSLRGLPV XAXXXXXXXXV B58 1369
    HBV 85 17 POL 545 GAKSVQHL XAXXXXXL B58 1370
    HBV 85 17 POL 545 GAKSVQHLESL XAXXXXXXXXL B58 1371
    HBV 75 15 ENV 134 GSSSGTVNPV XSXXXXXXXV B58 1372
    HBV 90 18 POL 735 GTDNSVVL XTXXXXXL B58 1373
    HBV 75 15 ENV 13 GTNLSVPNPL XTXXXXXXXL B58 1374
    HBV 80 16 POL 763 GTSFVYVPSAL XTXXXXXXXXL B58 1375
    HBV 85 17 POL 715 HTAELLAACF XTXXXXXXXF B58 1376
    HBV 100 20 NUC 52 HTALRQAI XTXXXXXI B58 1377
    HBV 95 19 NUC 52 HTALAQAIL XTXXXXXXL B58 5.0021 1378
    HBV 95 19 NUC 52 HTALROAILCW XTXXXXXXXXW B58 1379
    HBV 100 20 POL 149 HTLWKAGI XTXXXXXI B58 1380
    HBV 100 20 POL 149 HTLWKAGIL XTXXXXXXL B58 5.0033 1381
    HBV 100 20 POL 149 HTLWKAGILY XTXXXXXXXY B58 1.0542 * 1382
    HBV 90 18 NUC 105 ISQLTFGRETV XSXXXXXXXXV B58 1383
    HBV 85 17 POL 547 KSVQHLESL XSXXXXXXL B58 1384
    HBV 95 19 POL 574 KTKRWGYSL XTXXXXXXL B58 5.0034 1385
    HBV 85 17 POL 574 KTKRWGYSLNF XTXXXXXXXXF B58 1386
    HBV 90 18 POL 534 LAFSYMDDV XAXXXXXXV B58 20.0119 1387
    HBV 90 18 POL 534 LAFSYMDDVV XAXXXXXXXV B58 20.0257 1388
    HBV 90 18 POL 534 LAFSYMDDVVL XAXXXXXXXXL B58 1389
    HBV 95 19 POL 515 LAQFTSAI XAXXXXXI B58 1390
    HBV 95 19 POL 515 LAQFTSAICSV XAXXXXXXXXV B58 1391
    HBV 95 19 NUC 45 LSFLPSDF XSXXXXXF B58 1392
    HBV 95 19 NUC 45 LSFLPSDFF XSXXXXXXF B38 20.0123 1393
    HBV 95 19 POL 415 LSLDVSAAF XSXXXXXXF B58 1394
    HBV 95 19 POL 415 LSLDVSAAFY XSXXXXXXXY B58 2.0239 * 1395
    HBV 100 20 ENV 336 LSLLVPFV XSXXXXXV B58 1396
    HBV 100 20 ENV 336 LSLLVPFVQW XSXXXXXXXW B58 1397
    HBV 100 20 ENV 336 LSLLVPFVQWF XSXXXXXXXXF B58 1398
    HBV 95 19 X 53 LSLRGLPV XSXXXXXV B58 1399
    HBV 95 19 X 53 LSLRGLPVCAF XSXXXXXXXXF B58 1400
    HBV 95 19 POL 510 LSPFLLAQFP XSXXXXXXF B58 1401
    HBV 75 15 ENV 349 LSPTVWLSV XSXXXXXXV B58 1402
    HBV 75 15 ENV 349 LSPTVWLSVI XSXXXXXXXI B58 1403
    HBV 75 15 ENV 349 LSPTVWISVIW XSXXXXXXXXW B58 1404
    HBV 85 17 POL 742 LSRKYTSF XSXXXXXF B58 1405
    HBV 85 17 POL 742 LSRKYTSFPW XSXXXXXXXW B58 1406
    HBV 85 17 POL 742 LSRKVTSFPWL XSXXXXXXXXL B58 1407
    HBV 90 18 POL 408 LSSNLSWL XSXXXXXL B58 1408
    HBV 90 18 POL 408 LSSNLSWLSL XSXXXXXXXL B58 1409
    HBV 100 20 NUC 140 LSTLPETTV XSXXXXXXV B58 1410
    HBV 100 20 NUC 140 LSTLPETTVV XSXXXXXXXV B58 1411
    HBV 75 15 ENV 16 LSVPNPLGF XSXXXXXXF B58 1412
    HBV 100 20 POL 412 LSWLSLDV XSXXXXXV B58 1413
    HBV 75 15 POL 3 LSYQHFRKL XSXXXXXXL B58 1414
    HBV 75 15 POL 3 LSYQHFRKLL XSXXXXXXXL B58 1415
    HBV 75 15 POL 3 LSYQHFRKLLL XSXXXXXXXXL B58 1416
    HBV 95 19 NUC 108 LTFGRETV XTXXXXXV B58 1417
    HBV 75 15 NUC 137 LTFGRETVL XTXXXXXXL B58 1418
    HBV 75 15 NUC 137 LTFGRETVLEY XTXXXXXXXXY B58 1419
    HBV 90 18 ENV 189 LTIPQSLDSW XTXXXXXXXW B58 1420
    HBV 90 18 ENV 189 LTIPQSLDSWW XTXXXXXXXXW B58 1421
    HBV 90 18 POL 404 LTNLLSSNL XTXXXXXXL B58 1422
    HBV 90 18 POL 404 LTNLLSSNLSW XTXXXXXXXXW B58 1423
    HBV 80 16 ENV 185 LTRILTIPQSL XTXXXXXXXXL B58 1424
    HBV 85 17 POL 99 LTVNEKRRL XTXXXXXXL B58 1425
    HBV 75 15 X 103 MSTTDLEAY XSXXXXXXY B58 2.0126 * 1426
    HBV 75 15 X 103 MSTTDLEAYF XSXXXXXXXF B58 1427
    HBV 100 20 NUC 138 NAPILSTL XAXXXXXL B58 1428
    HBV 90 18 POL 738 NSVVLSRKY XSXXXXXXY B58 2.0123 1429
    HBV 100 20 POL 430 PAAMPHLL XAXXXXXL B58 1430
    HBV 85 17 POL 430 PAAMPHLLV XAXXXXXXV B58 1431
    HBV 90 18 POL 775 PADDPSRGRL XAXXXXXXXL B58 1432
    HBV 90 18 ENV 131 PAGGSSSGTV XAXXXXXXXV B58 1433
    HBV 95 19 POL 641 PALMPLYACI XAXXXXXXXI B58 5.0087 1434
    HBV 80 16 X 11 PARDVLCL XAXXXXXL B58 1435
    HBV 75 15 X 11 PARDVLCLRPV XAXXXXXXXXV B58 1436
    HBV 90 18 POL 355 PARVTGGV XAXXXXXV B58 1437
    HBV 90 18 POL 355 PARVTGGVF XAXXXXXXF B58 1438
    HBV 90 18 POL 355 PARVTGGVFL XAXXXXXXXL B58 1439
    HBV 90 18 POL 355 PARVTGGVFLV XAXXXXXXXXV B58 1440
    HBV 95 19 NUC 130 PAYRPPNAPI XAXXXXXXXI B58 5.0081 1441
    HBV 95 19 NUC 130 PAYRPPNAPIL XAXXXXXXXXL B58 1442
    HBV 90 18 POL 779 PSRGPLGL XSXXXXXL B58 1443
    HBV 75 15 POL 692 PTGWGLAI XTXXXXXI B58 1444
    HBV 85 17 POL 797 PTTGRTSL XTXXXXXL B58 1445
    HBV 85 17 POL 797 PTTGRTSLY XTXXXXXXY B58 1.0208 * 1446
    HBV 80 16 NUC 15 PTVQASKL XTXXXXXL B58 1447
    HBV 80 16 NUC 15 PTVQASKLCL XTXXXXXXXL B58 1448
    HBV 75 15 ENV 351 PTVWLSVI XTXXXXXI B58 1449
    HBV 75 15 ENV 351 PTVWLSVIW XTXXXXXXW B58 1450
    HBV 150 30 ENV 351 PTVWLSVIWM XTXXXXXXXM B58 1451
    HBV 95 19 POL 654 QAFTFSPTY XAXXXXXXY B58 20.0127 1452
    HBV 80 16 ENV 179 QAGFFLLTRIL XAXXXXXXXXL B58 1453
    HBV 90 18 NUC 57 QAILCWGEL XAXXXXXXL B58 1454
    HBV 180 36 NUC 57 QAILCWLELM XAXXXXXXXM B58 1456
    HBV 80 16 ENV 107 QAMQWNSTTF XAXXXXXXXF B58 1456
    HBV 80 16 NUC 18 QASKLCLGW XAXXXXXXW B58 1457
    HBV 80 16 NUC 18 QASKLCLGWL XAXXXXXXXL B58 1458
    HBV 75 15 NUC 18 QASKLCLGWLW XAXXXXXXXXW B58 1459
    HBV 90 18 ENV 193 QSLDSWWTSL XSXXXXXXXL B58 F126.63 1460
    HBV 90 18 POL 402 QSLTNLLSSNL XSXXXXXXXXL B58 1461
    HBV 95 19 POL 528 RAFPHCLAF XAXXXXXXF B58 20.0125 1462
    HBV 95 19 POL 528 RAFPHCLAFSY XAXXXXXXXXY B58 26.0550 * 1463
    HBV 90 18 POL 353 RTPARVTSGGV XTXXXXXXXV B58 1464
    HBV 90 18 POL 353 RTPARVTGGVF XTXXXXXXXXF B58 1465
    HBV 90 18 X 65 SAGPCALRF XAXXXXXXF B58 26.0152 1466
    HBV 95 19 POL 520 SAICSVVRRAF XAXXXXXXXXF B58 1467
    HBV 90 18 NUC 35 SALYREAL XAXXXXXL B58 1468
    HBV 100 20 POL 165 SASFCGSPY XAXXXXXXY B58 20.0117 * 1469
    HBV 100 20 POL 165 SASFCGSPYSW XAXXXXXXXXW B58 1470
    HBV 95 19 X 64 SSAGPCAL XSXXXXXL B58 1471
    HBV 90 18 X 64 SSAGPCALRF XSXXXXXXXF B58 26.0374 1472
    HBV 75 15 ENV 136 SSGTVNPV XSXXXXXV B58 1473
    HBV 90 18 POL 409 SSNLSWLSL XSXXXXXXL B58 1474
    HBV 90 18 POL 409 SSNLSWLSLDV XSXXXXXXXXV B58 1475
    HBV 75 15 ENV 135 SSSGTVNPV XSXXXXXXV B58 1476
    HBV 100 20 NUC 141 STLPETTV XTXXXXXV B58 1477
    HBV 100 20 NUC 141 STLPETTVV XTXXXXXXV B58 5.0024 1478
    HBV 75 15 X 104 STTDLEAY XTXXXXXY B58 1479
    HBV 75 15 X 104 STTDLEAYF XTXXXXXXF B58 1480
    HBV 85 17 POL 716 TAELLAACF XAXXXXXXF B58 1481
    HBV 95 19 NUC 53 TALRQAIL XAXXXXXL B58 1482
    HBV 95 19 NUC 53 TALRQAILCW XAXXXXXXXXW B58 1483
    HBV 80 16 NUC 33 TASALYREAL XAXXXXXXXL B58 1484
    HBV 95 19 POL 519 TSAICSVV XSXXXXXV B58 1485
    HBV 80 16 POL 764 TSFVYYPSAL XSXXXXXXXL B58 1486
    HBV 80 16 ENV 168 TSGFLGPL XSXXXXXL B58 1487
    HBV 75 15 ENV 168 TSQFLGPLL XSXXXXXXL B58 1488
    HBV 75 15 ENV 168 TSGFLGPLLV XSXXXXXXXV B58 1489
    HBV 75 15 ENV 168 TSGFLGPLLVL XSXXXXXXXXL B58 1490
    HBV 75 15 X 105 TTDLEAYF XTXXXXXF B58 1491
    HBV 85 17 POL 798 TTGRTSLY XTXXXXXY B58 26.0030 1492
    HBV 95 19 POL 37 VAEDLNLGNL XAXXXXXXXL B58 5.0089 1493
    HBV 100 20 POL 48 VSIPWTHKV XSXXXXXXV B58 1494
    HBV 85 19 POL 391 VSWPKFAV XSXXXXXV B58 1495
    HBV 95 19 POL 391 VSWPKFAVPNL XSXXXXXXXXL B58 1496
    HBV 100 20 POL 358 VTGGVFLV XTXXXXXV B58 1497
    HBV 85 17 ENV 66 WSPQAQGI XSXXXXXI B58 1498
    HBV 85 17 ENV 66 WSPQAQGIL XSXXXXXXL B58 1499
    HBV 100 20 POL 52 WTHKVGNF XTXXXXXF B58 1500
    HBV 95 19 POL 52 WTHKVGNFTGL XTXXXXXXXXL B58 1501
    HBV 80 16 POL 493 YSHPIILGF XSXXXXXXF B58 1502
    HBV 85 17 POL 580 YSLNFMGY XSXXXXXY B58 26.0032 1503
    HBV 75 15 POL 580 YSLNFMGYV XSXXXXXXV B58 1504
    HBV 75 15 POL 580 YSLNFMGYVI XSXXXXXXXI B58 1505
    HBV 85 17 POL 746 YTSFPWLL XTXXXXXL B58 1506
    189
  • TABLE XIV
    HBV B62 SUPER MOTIF
    SEQ ID
    Source Conservancy Freq Protein Position Sequence String Supermotif Peptide Filed NO:
    HBV 95 19 POL 521 AICSVVRRAF XIXXXXXXXF B62s 1507
    HBV 90 18 NUC 58 AILCWGELM XIXXXXXXM B62s 1508
    HBV 95 19 POL 642 ALMPLYACI XLXXXXXXI B62s 3.0012 * 1509
    HBV 95 19 NUC 54 ALRQAILCW XLXXXXXXW B62s 1510
    HBV 80 16 ENV 106 AMQWMSTTF XMXXXXXXF B62s 1511
    HBV 95 19 POL 633 APFTQCGY XPXXXXXY B62s 19.0013 1512
    HBV 95 19 POL 516 AQFTSAICSV XQXXXXXXXV B62s 1513
    HBV 95 19 POL 516 AQPTSAICSVV XQXXXXXXXXV B62s 1514
    HBV 100 20 ENV 312 CIPIPSSW XIXXXXXW B62s 1515
    HBV 100 20 ENV 312 CIPIPSSWAF XIXXXXXXXF B62s 1516
    HBV 90 18 POL 533 CLAFSYMDDV XLXXXXXXXV B62s 1.0559 1517
    HBV 90 18 POL 533 CLAFSYMDDVV XLXXXXXXXXV B62s 1518
    HBV 85 17 NUC 23 CLGWLWGM XLXXXXXM B62s 1519
    HBV 85 17 NUC 23 CLGWLWGMDI XLXXXXXXXI B62s 2.0229 1520
    HBV 95 19 ENV 253 CLIFLLVLLDY XLXXXXXXXXY B62s 26.0548 1521
    HBV 95 19 ENV 239 CLRRFIIF XLXXXXXF B62s 1522
    HBV 75 15 ENV 239 CLRRFIIFLF XLXXXXXXXF B62s 1523
    HBV 75 15 ENV 239 CLRRFIIFLFI XLXXXXXXXXI B62s Chisari 1524
    HBV 90 18 NUC 107 CLTFGRETV XLXXXXXXV B62s 1.0160 1525
    HBV 80 16 X 7 CQLDPARDV XQXXXXXXV B62s 1526
    HBV 85 17 POL 622 CQRVGLLGF XIXXXXXF B62s 1527
    HBV 90 18 NUC 31 DIDPYKEF XIXXXXXF B62s 1528
    HBV 85 17 NUC 29 DLLDTASALY XLXXXXXXXY B62s 1.0519 * 1529
    HBV 95 19 POL 40 DUNLGNLNV XLXXXXXXV B62s 1.0164 1530
    HBV 95 19 POL 40 DLNLGNLNVSI XLXXXXXXXXI B62s 1531
    HBV 80 16 ENV 122 DPRVRGLY XPXXXXXY B62s 1532
    HBV 95 19 X 14 DVLCLRPV XVXXXXXV B62s 1533
    HBV 90 18 POL 541 DVVLGAKSV XVXXXXXXV B62s 1.0190 1534
    HBV 95 19 NUC 43 ELLSFLPSDF XLXXXXXXXF B62s 1535
    HBV 95 19 NUC 43 ELLSFLPSDFF XLXXXXXXXXF B62s 1536
    HBV 80 16 ENV 248 FILLLCLI XIXXXXXI B62s Chisari 1537
    HBV 80 16 ENV 248 FILLLCLIF XIXXXXXXF B62s 1538
    HBV 80 16 ENV 246 FLFILLLCLI XLXXXXXXXI B62s 3.0206 1539
    HBV 80 16 ENV 246 FLFILLLCLIF XLXXXXXXXXF B62s 1540
    HBV 95 19 POL 513 FLLAQPTSAI XLXXXXXXXI B62s 1147.13 * 1541
    HBV 80 16 ENV 183 FLLTRILTI XLXXXXXXI B62s 3.0005 * 1542
    HBV 95 19 ENV 256 FLLVLLDY XLXXXXXY B62s 26.0027 1543
    HBV 95 19 ENV 256 FLLVLLDYQGM XLXXXXXXXXM B62s 1544
    HBV 75 15 ENV 130 FPAGGSSSGTV XPXXXXXXXXV B62s 1545
    HBV 85 17 ENV 14 FPQHQLDPAF XPXXXXXXXF B62s 20.0274 1546
    HBV 95 19 POL 530 FPHCLAFSY XPXXXXXXY B62s 15.0037 * 1547
    HBV 95 19 POL 530 FPHCLAFSYM XPXXXXXXXM B62s 16.0217 * 1548
    HBV 75 15 POL 749 FPWLLGCAANW XPXXXXXXXXW B62s 1549
    HBV 95 19 ENV 346 FVGLSPTV XYXXXXXV B62s 1550
    HBV 95 19 ENV 346 FVGLSPTVW XVXXXXXXW B62s 1551
    HBV 90 18 X 132 FVLGGCRHKLV XVXXXXXXXXV B62s 1552
    HBV 95 19 POL 627 GLLGFAAPF XLXXXXXXF B62s 20.0124 1553
    HBV 95 19 POL 509 GLSPFLLAQF XLXXXXXXXF B62s 1554
    HBV 75 15 ENV 348 GLSPTVWLSV XLXXXXXXXV B62s 1.0518 * 1555
    HBV 75 15 ENV 348 GLSPTVWLSVI XLXXXXXXXXI B62s Chisari 1556
    HBV 85 17 NUC 29 GMDIDPYKEF XMXXXXXXXF B62s 26.0372 1557
    HBV 95 19 ENV 173 GPLLVLQAGF XPXXXXXXXF B62s 15.0212 1558
    HBV 95 19 ENV 173 GPLLVLQAGFF XPXXXXXXXXF B62s 26.0556 1559
    HBV 95 19 NUC 123 GVWIRTPPAY XVXXXXXXXY B62s 1.0525 1560
    HBV 75 15 POL 569 HLNPNKTKRW XLXXXXXXXW B62s 1561
    HBV 90 18 X 52 HLSLRGLPV XLXXXXXXV B62s 1.0212 1562
    HBV 80 16 POL 491 HLYSHPII XLXXXXXI B62s 17.0256 1563
    HBV 80 16 POL 491 HLYSHPIILGF XLXXXXXXXXF B62s 1564
    HBV 85 17 POL 429 HPAAMPHLLV XPXXXXXXXV B62s 20.0273 * 1565
    HBV 80 16 POL 495 HPIILGFRKI XPXXXXXXXI B62s 1566
    HBV 80 16 POL 497 IILGFRKI XIXXXXXI B62s 17.0124 * 1567
    HBV 80 16 POL 497 IILGFRKIPM XIXXXXXXXM B62s 1568
    HBV 90 18 NUC 59 ILCWGELM XLXXXXXM B62s 1569
    HBV 80 16 POL 498 ILGFRKIPM XLXXXXXXM B62s 3.0016 1570
    HBV 100 20 ENV 249 ILLLCLIF XLXXXXXF B62s 1571
    HBV 100 20 ENV 249 ILLLCLIFLLV XLXXXXXXXXV B62s Chisari 1572
    HBV 80 16 POL 760 ILRGTSFV XLXXXXXV B62s 1573
    HBV 80 16 POL 760 ILRGTSFVY XLXXXXXXY B62s 1.0205 * 1574
    HBV 80 16 POL 760 ILRGTSFVYV XLXXXXXXXV B62s 1.0573 * 1575
    HBV 100 20 NUC 139 ILSTLPETTV XLXXXXXXXV B62s 1.0526 1576
    HBV 100 20 NUC 139 ILSTLPETTVV XLXXXXXXXXV B62s * 1577
    HBV 90 18 ENV 188 ILTIPQSLDSW XLXXXXXXXXW B62s 1578
    HBV 100 20 ENV 313 IPIPSSWAF XPXXXXXXF B62s 15.0032 * 1579
    HBV 80 16 POL 504 IPMGVGLSPF XPXXXXXXXF B62s 1580
    HBV 90 18 ENV 191 IPQSLDSW XPXXXXXW B62s 19.0004 1581
    HBV 90 18 ENV 191 IPQSLDSWW XPXXXXXXW B62s 15.0030 * 1582
    HBV 100 20 POL 50 IPWTHKVGNF XPXXXXXXXF B62s 15.0209 1583
    HBV 90 18 POL 625 IVGLLGFAAPF XVXXXXXXXXF B62s 1584
    HBV 80 16 POL 503 KIPMGVGLSPF XIXXXXXXXXF B62s 1585
    HBV 85 17 NUC 21 KLCLGWLW XLXXXXXW B62s 1586
    HBV 85 17 NUC 21 KLCLGWLWGM XLXXXXXXXM B62s 3.0209 1587
    HBV 95 19 POL 489 KLHLYSHPI XLXXXXXXI B62s 3.0009 1588
    HBV 80 16 POL 489 KLHLYSHPII XLXXXXXXXI B62s 1589
    HBV 75 15 POL 108 KLIMPARF XLXXXXXF B62s 1590
    HBV 75 15 POL 108 KLIMPARFY XLXXXXXXY B62s 1.0171 1591
    HBV 80 16 POL 610 KLPVNRPI XLXXXXXI B62s 1592
    HBV 80 16 POL 610 KLPVNRPIDW XLXXXXXXXW B62s 1593
    HBV 95 19 POL 653 KQAFTFSPTY XQXXXXXXXY B62s 20.0256 1594
    HBV 95 19 POL 55 KVGNFTGLY XVXXXXXXY B62s 1.0166 1595
    HBV 95 19 ENV 254 LIFLLVLLDY XIXXXXXXXY B62s 1.0899 1596
    HBV 100 20 POL 109 LIMPARFY XIXXXXXY B62s 26.0028 1597
    HBV 95 19 POL 514 LLAQPTSAI XLXXXXXXI B62s 3.0010 1598
    HBV 100 20 ENV 251 LLCLIFLLV XLXXXXXXV B62s 1.0835 1599
    HBV 85 17 NUC 30 LLDTASALY XLXXXXXXY B62s 1.0155 1600
    HBV 90 18 ENV 260 LLDYQGMLPV XLXXXXXXXV B62s 1.0516 1601
    HBV 80 16 POL 752 LLGCAANW XLXXXXXW B62s 1602
    HBV 80 16 POL 752 LLGCAANWI XLXXXXXXI B62s 3.0013 1603
    HBV 95 19 POL 628 LLGFAAPF XLXXXXXF B62s 1604
    HBV 75 15 ENV 63 LLGWSPQAQGI XLXXXXXXXXI B62s 1605
    HBV 100 20 ENV 250 LLLCLIFLLV XLXXXXXXXV B62s 1.0897 1606
    HBV 100 20 ENV 378 LLPIFFCLW XLXXXXXXW B62s 1607
    HBV 100 20 ENV 378 LLPIFFCLWV XLXXXXXXXV B62s 1.0904 1608
    HBV 100 20 ENV 378 LLPIFFCLWVY XLXXXXXXXXY B62s 26.0549 1609
    HBV 95 19 NUC 44 LLSFLFSDF XLXXXXXXF B62s 1610
    HBV 95 19 NUC 44 LLSFLPSDFF XLXXXXXXXF B62s 1611
    HBV 90 18 POL 407 LLSSNLSW XLXXXXXW B62s 1612
    HBV 80 16 ENV 184 LLTRILTI XLXXXXXI B62s Chisari 1613
    HBV 80 16 POL 436 LLVGSSGL XLXXXXXL B62s 1614
    HBV 95 19 ENV 257 LLVLLDYQGM XLXXXXXXXM B62s 3.0207 1615
    HBV 95 19 ENV 175 LLVLQAGF XLXXXXXF B62s 1616
    HBV 95 19 ENV 175 LLVLQAGFF XLXXXXXXF B62s 20.0121 1617
    HBV 100 20 ENV 338 LLVPFVQW XLXXXXXW B62s 1618
    HBV 100 20 ENV 338 LLVPFVQWF XLXXXXXXF B62s 1619
    HBV 95 19 ENV 338 LLVPFVQWFV XLXXXXXXXV B62s 1.0930 1620
    HBV 85 17 NUC 100 LLWFHISCLTF XLXXXXXXXXF B62s 1621
    HBV 95 19 POL 643 LMPLYACI XMXXXXXI B62s 17.0130 1622
    HBV 100 20 ENV 379 LPIFFCLW XPXXXXXW B62s 19.0007 1623
    HBV 100 20 ENV 379 LPIFFCLWV XPXXXXXXV B62s 15.0034 1624
    HBV 100 20 ENV 379 LPIFFCLWVY XPXXXXXXXY B62s 15.0215 1625
    HBV 100 20 ENV 379 LPIFFCLWVYI XPXXXXXXXXI B62s 26.0558 1626
    HBV 100 20 POL 123 LPLDKGIKPY XPXXXXXXXY B62s 15.0210 1627
    HBV 100 20 POL 123 LPLDKGIKPYY XPXXXXXXXXY B62s 26.0560 1628
    HBV 80 16 POL 611 LPVNRPIDW XPXXXXXXW B62s 1629
    HBV 80 16 POL 611 LPVNRPIDWKV XPXXXXXXXXV B62s 1630
    HBV 80 16 ENV 178 LQAGFFLLTRI XQXXXXXXXXI B62s 1631
    HBV 95 19 ENV 258 LVLLDYQGM XVXXXXXXM B62s 3.0034 1632
    HBV 95 19 ENV 176 LVLDAGFF XVXXXXXF B62s 1633
    HBV 100 20 ENV 339 LVPFVQWF XVXXXXXF B62s 1634
    HBV 95 19 ENV 339 LVPFVQWFV XVXXXXXXV B62s 1.0877 1635
    HBV 90 18 NUC 119 LVSFGVWI XVXXXXXI B62s Chisari 1636
    HBV 100 20 POL 377 LVVDFSQF XVXXXXXF B62s 1637
    HBV 85 17 ENV 0 MMWYWGPSLY XMXXXXXXXY B62s 1039.01 1638
    HBV 100 20 POL 1 MPLSYQHF XPXXXXXF B62s 19.0010 1639
    HBV 80 16 ENV 109 MQWNSTTF XQXXXXXF B62s 1640
    HBV 95 19 POL 42 NLGNLNVSI XLXXXXXXI B62s 3.0008 1641
    HBV 95 19 POL 42 NLGNLNVSIPW XLXXXXXXXXW B62s 1642
    HBV 90 18 POL 406 NLLSSNLSW XLXXXXXXW B62s 1643
    HBV 95 19 POL 45 NLNVSIPW XLXXXXXW B62s 1644
    HBV 75 15 ENV 15 NLSVPNPLGF XLXXXXXXXF B62s 1645
    HBV 90 18 POL 411 NLSWLSLDV XLXXXXXXV B62s 1.0185 1646
    HBV 75 15 POL 571 NPNKTKRW XPXXXXXW B62s 1647
    HBV 75 15 POL 571 NPNKTKRWGY XPXXXXXXXY B62s 1648
    HBV 100 20 POL 47 NVSIPWTHKV XVXXXXXXXV B62s 1.0532 1649
    HBV 85 17 POL 616 PIDWKVCQRI XIXXXXXXXI B62s Chisari 1650
    HBV 85 17 POL 616 PIDWKCQRIV XIXXXXXXXXV B62s 1651
    HBV 100 20 ENV 380 PIFFCLWV XIXXXXXV B62s 1652
    HBV 100 20 ENV 380 PIFFCLWVY XIXXXXXXY B62s 1.0843 1653
    HBV 100 20 ENV 380 PIFFCLWVYI XIXXXXXXXI B62s 20.0258 1654
    HBV 80 16 POL 496 PIILGFRKI XIXXXXXXI B62s 927.48 1655
    HBV 80 16 POL 496 PIILGFRKIPM XIXXXXXXXXM B62s 1656
    HBV 100 20 NUC 136 PILSTLPETTV XIXXXXXXXXV B62s Chisari 1657
    HBV 100 20 ENV 314 PIPSSWAF XIXXXXXF B62s 1658
    HBV 100 20 POL 124 PLDKGIKPY XLXXXXXXY B62s 1.0174 1659
    HBV 100 20 POL 124 PLDKGIKPYY XLXXXXXXXY B62s 1.0541 1660
    HBV 20 20 ENV 377 PLLPIFFCLW XLXXXXXXXW B62s 1661
    HBV 100 20 ENV 377 PLLPIFFCLWV XLXXXXXXXXV B62s 1662
    HBV 95 19 ENV 174 PLLVLQAGF XLXXXXXXF B62s 1663
    HBV 95 19 ENV 174 PLLVLQAGFF XLXXXXXXXF B62s 1664
    HBV 80 16 POL 505 PMGVGLSPF XMXXXXXXF B62s 1665
    HBV 95 19 NUC 129 PPAYRPPNAPI XPXXXXXXXXI B62s 26.0563 1666
    HBV 85 17 ENV 58 PPHGGLLGW XPXXXXXXW B62s 20.0141 1667
    HBV 80 16 ENV 106 PQAMQWNSTT XQXXXXXXXXF B62s 1668
    HBV 90 18 ENV 192 PQSLDSWW XQXXXXXW B62s 1669
    HBV 85 17 POL 612 PVNRPIDW XVXXXXXW B62s 1670
    HBV 85 17 POL 612 PVNRPIDWKV XVXXXXXXXV B62s 1.0566 1671
    HBV 80 16 X 8 QLDPARDV XLXXXXXV B62s Chisari 1672
    HBV 95 19 POL 665 QVFADATPTGW XVXXXXXXXXW B62s 1673
    HBV 90 18 POL 624 RIVGLLGF XIXXXXXF B62s 1674
    HBV 75 15 POL 106 RLKLIMPARF XLXXXXXXXF B62s 1675
    HBV 75 15 POL 106 RLKLIMPARFY XLXXXXXXXXY B62s 1676
    HBV 95 19 POL 376 RLVVDFSQF XLXXXXXXF B62s 20.0122 1677
    HBV 80 16 POL 615 RPIDWKVCQRI XPXXXXXXXXI B62s 1678
    HBV 90 18 NUC 56 RQAILCWGELM XQXXXXXXXXM B62s 1679
    HBV 90 18 NUC 98 RQLLWFHI XQXXXXXI B62s 1680
    HBV 75 15 POL 818 RVHFASPLHV XVXXXXXXXV B62s 1.0576 1681
    HBV 100 20 POL 357 RVTGGVFLV XVXXXXXXV B62s 1.0181 1682
    HBV 100 20 POL 49 SIPWTHKV XIXXXXXV B62s 1683
    HBV 100 20 POL 49 SIPWTHKVGNF XIXXXXXXXXF B62s 1684
    HBV 95 19 ENV 194 SLDSWWTSLNF XLXXXXXXXXF B62s 1685
    HBV 95 19 POL 416 SLDVSAAF XLXXXXXF B62s 1686
    HBV 95 19 POL 416 SLDVSAAFY XLXXXXXXY B62s 1.0186 * 1687
    HBV 100 20 ENV 337 SLLVPFVQW XLXXXXXXW B62s 1688
    HBV 100 20 ENV 337 SLLVPFVQWF XLXXXXXXXF B62s 1689
    HBV 95 19 ENV 337 SLLVPFVQWFV XLXXXXXXXXV B62s 1690
    HBV 75 15 POL 581 SLNFMGYV XLXXXXXV B62s 1691
    HBV 75 15 POL 581 SLNFMGYVI XLXXXXXXI B62s 3.0011 1692
    HBV 95 19 X 54 SLRGLPVCAF XLXXXXXXXF B62s 20.0259 1693
    HBV 95 19 POL 511 SPFLLAQF XPXXXXXF B62s 19.0012 * 1694
    HBV 100 20 NUC 49 SPHHTALRQAI XPXXXXXXXXI B62s 26.0567 * 1695
    HBV 75 15 ENV 350 SPTVWLSV XPXXXXXV B62s 1696
    HBV 75 15 ENV 330 SPTVWLSVI XPXXXXXXI B62s 1308.16 1697
    HBV 75 15 ENV 350 SPTVWLSVIW XPXXXXXXXW B62s 1308.17 1698
    HBV 75 15 ENV 350 SPTVWLSVIWM XPXXXXXXXXM B62s 1699
    HBV 75 15 ENV 17 SVPNPLGF XVXXXXXF B62s 1700
    HBV 80 16 ENV 330 SVRFSWLSLLV XVXXXXXXXXV B62s 1701
    HBV 90 18 POL 739 SVVLSRKY XVXXXXXY B62s 26.0029 1702
    HBV 85 17 POL 739 SVVLSRKYTSF XVXXXXXXXXF B62s 1703
    HBV 90 18 ENV 190 TIPQSLDSW XIXXXXXXW B62s 1704
    HBV 90 18 ENV 190 TIPQSLDSWW XIXXXXXXXW B62s 1705
    HBV 100 20 NUC 142 TLPETTVV XLXXXXXV B62s 1706
    HBV 100 20 POL 150 TLWKAGILY XLXXXXXXY B62s 1.0177 * 1707
    HBV 90 18 POL 354 TPARVTGGV XPXXXXXXV B62s 15.0033 * 1708
    HBV 90 18 POL 354 TPARVTGGVF XPXXXXXXXF B62s 15.0214 * 1709
    HBV 75 15 ENV 57 TPPHGGLLGW XPXXXXXXXW B62s 1308.04 1710
    HBV 75 15 POL 691 TPTGWGLAI XPXXXXXXI B62s 1711
    HBV 95 19 POL 636 TQCGYPALM XQXXXXXXM B62s 1712
    HBV 80 16 NUC 16 TVQASKLCLGW XVXXXXXXXXW B62s 1713
    HBV 75 15 ENV 352 TVWLSVIW XVXXXXXW B62s 1714
    HBV 75 15 ENV 352 TVWLSVIWM XVXXXXXXM B62s 3.0035 1715
    HBV 90 18 X 133 VLGGCRHKLV XLXXXXXXXV B62s 1.0589 1716
    HBV 95 19 ENV 259 VLLDYQGM XLXXXXXM B62s 17.0107 1717
    HBV 90 18 ENV 259 VLLDYQGMLPV XLXXXXXXXXV B62s 1147.14 * 1718
    HBV 85 17 POL 741 VLSRKYTSF XLXXXXXXF B62s 1719
    HBV 85 17 POL 741 VLSRKYTSFPW XLXXXXXXXXW B62s 1720
    HBV 95 19 ENV 340 VPFVQWFV XPXXXXXV B62s 19.0006 * 1721
    HBV 80 16 NUC 17 VQASKLCLGW XQXXXXXXXW B62s 1722
    HBV 95 19 ENV 343 VQWFVGLSPTV XQXXXXXXXXV B62s 1723
    HBV 90 18 POL 542 VVGAKSV XVXXXXXV B62s 1724
    HBV 80 16 POL 759 WILRGTSF XIXXXXXXF B62s 1725
    HBV 80 16 POL 759 WILRGTSFV XIXXXXXXV B62s 1.0204 * 1726
    HBV 80 16 POL 759 WILRGTSFVY XIXXXXXXXY B62s 1.0572 1727
    HBV 80 16 POL 759 WILRGTSFVYV XIXXXXXXXXV B62s 1728
    HBV 95 19 NUC 125 WIRTPPAY XIXXXXXY B62s 26.0031 1729
    HBV 80 16 POL 751 WLLGCAANW XLXXXXXXW B62s 1730
    HBV 80 16 POL 751 WLLGCAANWI XLXXXXXXXI B62s Chisari 1731
    HBV 95 19 POL 414 WLSLDVSAAF XLXXXXXXXF B62s 1732
    HBV 95 19 POL 414 WLSLDVSAAFY XLXXXXXXXXY B62s 26.0551 1733
    HBV 100 20 ENV 335 WLSLLVPF XLXXXXXF B62s 1734
    HBV 100 20 ENV 335 WLSLLVPFV XLXXXXXXV B62s 1.0838 * 1735
    HBV 100 20 ENV 335 WLSLLVPFVQW XLXXXXXXXXW B62s 1736
    HBV 85 17 NUC 26 WLWGMDIDPY XLXXXXXXXY B62s 1.0774 * 1737
    HBV 95 19 ENV 237 WMCLRRFI XMXXXXXI B62s 1738
    HBV 95 19 ENV 237 WMCLRRFI XMXXXXXXI B62s 3.0031 * 1739
    HBV 95 19 ENV 237 VMCLRRFIIF XMXXXXXXXF B62s 20.0266 1740
    HBV 85 17 ENV 359 WMMWYWGPSL XMXXXXXXXL B62s 1.0901 * 1741
    HBV 100 20 POL 147 YLHTLWKAGI XLXXXXXXXI B62s 7.0066 1742
    HBV 100 20 POL 122 YLPLDKGI XLXXXXXI B62s 1743
    HBV 100 20 POL 122 YLPLDKGIKPY XLXXXXXXXXY B62s 26.0553 1744
    HBV 90 18 NUC 118 YLVSFGVW XLXXXXXW B62s 1745
    HBV 90 18 NUC 118 YLVSFGVWI XLXXXXXXI B62s 3.0007 * 1746
    HBV 95 19 POL 640 YPALMPLY XPXXXXXY B62s 19.0014 * 1747
    HBV 95 19 POL 640 YPALMPLYACI XPXXXXXXXXI B62s 26.0570 1748
    242
  • TABLE XV
    HBV A01 Motif (With Binding Information)
    1st SEQ ID
    Conservancy Frequency Protein Pos Sequence P2 C-term Peptide Filed A*0101 NO:
    80 16 ENV 119 AMQWNSTTF M F 1749
    90 18 POL 737 DNSVVLSRKY N Y 20.0255 0.0001 1750
    95 19 POL 631 FAAPFTQCGY A Y 20.0254 * 0.0680 1751
    85 17 POL 590 GYSLNFMGY Y Y 2.0058 1752
    100 20 POL 149 HTLWKAGILY T Y 1069.04 * 0.1100 1753
    95 19 POL 653 KQAFTFSPTY Q Y 20.0256 0.0001 1754
    85 17 NUC 30 LLDTASALY L Y 1069.01 * 12.0000 1755
    95 19 POL 415 LSLDVSAAFY S Y 1090.07 * 0.0150 1756
    85 17 ENV 360 MMWYWGPSLY M Y 1039.01 * 0.0810 1757
    75 15 X 103 MSTTDLEAY S Y 2.0126 * 0.8500 1758
    90 18 POL 738 NSVVLSRKY S Y 2.0123 0.0005 1759
    100 20 POL 124 PLDKGIKPY L Y 1147.12 * 1760
    100 20 POL 124 PLDKGIKPYY L Y 1069.03 * 0.1700 1761
    85 17 POL 797 PTTGRTSLY T Y 1090.09 * 0.2100 1762
    100 20 POL 165 SASFCGSPY A Y 1763
    95 19 POL 416 SLDVSAAFY L Y 1069.02 * 5.2000 1764
    16
  • TABLE XVI
    HBV A03 and A11 Motif (With binding information)
    Conser- Fre- 1st Super C- SEQ ID
    vancy quency Protein Pos Sequence Motif Motif P2 term Peptide Filed A*0301 A*1101 NO:
    85 17 POL 721 AACFARSR A03/A11 A03 A R 26.0003 0.0004 0.0003 1785
    95 19 POL 643 AAPFTQCGY A03/A11 A03 A Y 1766
    95 19 POL 540 AFPHCLAFSY A03/A11 A03 F Y 1767
    95 19 X 62 AFSSAGPCA A03/A11 A03 F A 1768
    95 19 POL 666 AFTFSPTYK A03/A11 A03 F K 20.0130 * 0.2600 0.0400 1769
    95 19 POL 666 AFTFSPTYKA A03/A11 A03 F A 1770
    95 19 POL 18 AGPLEEEPR A03/A11 A03 G R 20.0265 0.0004 0.0002 1771
    95 19 POL 521 AICSVVRR A03/A11 A03 I R 26.0004 −0.0002 0.0003 1772
    95 19 POL 532 AICSVVRRAF A03/A11 A03 I F 1773
    90 18 POL 772 ALNPADDPSR A03/A11 A03 L R 1.1090 0.0003 0.0001 1774
    85 17 X 70 ALRFTSAR A03/A11 A03 L R 26.0005 0.0047 0.0009 1775
    80 16 ENV 119 AMQWNSTTF A03/A11 A03 M F 1776
    80 16 ENV 119 AMQWNSTTF A03/    A03 M F 1777
    80 16 ENV 119 AMQWNSTTFH A03/A11 A03 M H 1778
    80 16 POL 822 ASPLHAWR A03/A11 A03 S R 1779
    75 15 ENV 84 ASTNRQSGR A03/A11 A03 S R 1150.60 0.0009 0.0002 1780
    80 16 POL 755 CAANWILR A03/A11 A03 A R 1781
    85 17 X 69 CALRFTSAR A03/A11 A03 A R 26.0149 * 0.0034 0.0230 1782
    85 17 POL 734 CFAPSRSGA A03/A11 A03 F A 1783
    75 15 POL 618 CFPKLPVNR A03/A11 A03 F R 1784
    95 19 POL 649 CGYPALMPLY A03/A11 A03 G Y 1785
    100 20 ENV 323 CIPIPSSWAF A03/A11 A03 I F 1786
    90 18 X 17 CLRPVGAESR A03/A11 A03 L P 1.1093 0.0011 0.0001 1787
    75 15 ENV 239 CLRRFIIFLF A03/A11 A03 L F 1788
    100 20 NUC 48 CSPHHTALR A03/A11 A03 S R 5.0055 * 0.0029 0.0001 1789
    95 19 POL 534 CSVVRRAFPH A03/A11 A03 S H 1790
    85 17 NUC 58 DLLDTASALY A03/A11 A03 L Y 1.0519 * 0.0001 0.0001 1791
    85 17 NUC 29 DLLDTASALYR A03/A11 A03 L R 26.0530 0.0042 −0.0003 1792
    95 19 ENV 207 DSWWTSLNF A03/A11 A03 S F 20.0120 0.0006 0.0002 1793
    85 17 NUC 32 DTASALYR A03/A11 A03 T R 26.0006 0.0004 −0.0002 1794
    95 19 POL 17 EAGPLEEELPR A03/A11 A03 A R 26.0531 −0.0009 −0.0003 1795
    90 18 POL 718 ELLAACFAR A03/A11 A03 L R 1.0988 0.0002 0.0004 1796
    85 17 POL 718 ELLAACFARSR A03/A11 A03 L R 26.0532 0.0062 0.0016 1797
    95 19 NUC 43 ELLSFLPSDF A03/A11 A03 L F 1798
    95 19 NUC 72 ESPEHCSPH A03/A11 A03 S H 1799
    95 19 NUC 72 ESPEHCSPHH A03/A11 A03 S H 1800
    95 19 NUC 174 ETTVVRRR A03/A11 A03 T R 26.0007 0.0003 −0.0002 1801
    80 16 NUC 174 ETTVVRRRGR A03/A11 A03 T R 1.1073 0.0003 0.0001 1802
    95 19 POL 642 FAAPFTQCGY  A01/A03/ A03 A Y 20.0254 * 1803
    80 16 POL 821 FASPLHVAWR A03/A11 A03 A R 1804
    90 18 ENV 24 FFPDHQLDPA A03/A11 A03 F A 1805
    75 15 NUC 139 FRRETVLEY A03/A11 A03 G Y 1806
    75 15 POL 255 FGVEPSGSGH A03/A11 A03 G H 1807
    80 16 ENV 248 FLLLICLIF A03/A11 A03 I F 1808
    90 18 X 63 FSSAGPCALR A03/A11 A03 S R 1809
    100 20 ENV 344 FSWLSUVPF A03/A11 A03 S F 20.0263 0.0004 0.0002 1810
    95 19 POL 656 FTFSPTYK A03/A11 A03 T K 1147.19 * 0.0100 0.0100 1811
    95 19 POL 667 FTFSPTYKAF A03/A11 A03 T F 20.0263 0.0004 0.0006 1812
    95 19 POL 518 FTSAICSVVR A03/A11 A03 T R 1.1085 0.0003 0.0003 1813
    95 19 POL 518 FTSAICSVVRR A03/A11 A03 T R 26.0533 0.0065 0.0092 1814
    90 18 X 132 FVLGGCRHK A03/A11 A03 V K 1090.03 * 0.0430 0.0090 1815
    80 16 POL 765 GCAANWILR A03/A11 A03 C R 1816
    75 15 POL 567 GIHLNPNK A03/A11 A03 I K 1817
    75 15 POL 567 GIHLNPNKTK A03/A11 A03 I K 1.0583 0.0025 0.0011 1818
    75 15 POL 567 GIHLNPNKTKR A03/A11 A03 I R 1819
    95 19 POL 638 GLLGFAAPF A03/A11 A03 L F 20.0124 0.0006 0.0002 1820
    95 19 POL 520 GLSPFLLAQF A03/A11 A03 L F 1821
    85 17 NUC 29 GMDIDPYK A03/AAA A03 M K 26.0009 0.0006 0.0004 1822
    85 17 NUC 29 GMDIDPYKEF A03/A24 A03 M F 26.0372 −0.0003 −0.0002 1823
    90 16 POL 735 GTDNSVVLSR A03/A11 A03 T R 1090.04 * 0.0010 0.0420 1824
    90 18 POL 735 GTDNSVVLSRK A03/A11 A03 T K 1147.17 * 0.0140 0.5800 1825
    80 16 POL 256 GVEPSGSGH A03/A11 A03 V H 1826
    100 20 POL 372 GVFLVDKNPH A03/A11 A03 V H 1827
    95 19 NUC 152 GVWIRTPPAY A03/A11 A03 V Y 1.0525 0.0047 0.0002 1828
    95 19 NUC 123 GVWIRTPPAYR A03/A11 A03 V R 26.0535 * 0.1900 0.1700 1829
    100 20 NUC 76 HCSPHHTALR A03/A11 A03 C R 1830
    80 16 POL 831 HFASPLHVA A03/A11 A03 F A 1831
    90 18 NUC 104 HISCLTFGR A03/A11 A03 I R 1069.18 * 0.0160 0.0065 1832
    75 15 POL 569 HLNPNKTK A03/A11 A03 L K 1833
    75 15 POL 569 HLNPNKTKR A03/A11 A03 L R 1.0983 0.0025 0.0001 1834
    85 17 POL 726 HTAELLAACF A03/A11 A03 T F 1835
    100 20 POL 149 HTLWKAGILYK A03/A11 A03 T K 1147.16 * 0.5400 0.4400 1836
    95 19 POL 533 ICSVVRRAF A03/A11 A03 C F 1837
    95 19 ENV 266 IFLLVLLDY A03/A11 A03 F Y 1838
    80 16 POL 771 ILRGTSFVY A03/A11 A03 L Y 1.0205 * 0.0440 0.0002 1839
    90 18 NUC 105 ISCLTFGR A03/A11 A03 S R 26.0010 0.0004 0.0002 1840
    100 20 POL 153 KAGILYKR A03/A11 A03 A R 26.0011 0.0002 −0.0002 1841
    75 15 POL 108 KLIMPARFY A03/A11 A03 L Y 1.0171 1842
    80 16 POL 610 KLPVNRPIDWK A03/A11 A03 L K 1843
    75 15 X 130 IWFVLGGCR A03/A11 A03 V R 1.0993 * 0.0420 0.0820 1844
    75 15 X 130 KVFVLGGCRH A03/A11 A03 V H 1845
    95 19 POL 55 KVGNFTGLY A03/A11 A03 V Y 1142.05 * 0.2100 0.0170 1846
    85 17 POL 720 LAACFAASR A03/A11 A03 A R 20.0129 0.0058 0.0065 1847
    100 20 POL 125 LDKGIKPYY A03/A11 A03 D Y 1848
    95 19 ENV 206 LDSWWTSLNF A03/A11 A03 D F 1849
    85 17 NUC 60 LDTASALYR A03/A11 A03 D R 26.0151 0.0004 −0.0002 1850
    95 19 POL 428 LDVSAAFYH A03/A11 A03 D H 1851
    80 16 ENV 247 LFILLLCLIF A03/A11 A03 F F 1852
    80 16 ENV 247 LFILLLCLIF A03/A24 A03 F F 1853
    80 16 POL 764 LGCAANWILR A03/A11 A03 G R 1854
    75 15 POL 577 LGIHLNPNK A03/A11 A03 G K 1855
    95 19 ENV 265 LIFLLVLLDY A03/A11 A03 I Y 1.0899 0.0022 0.0004 1856
    90 18 POL 719 LLAACFAR A03/A11 A03 L R 26.0012 0.0024 0.0003 1857
    85 17 POL 719 LLAACFARSR A03/A11 A03 L R 1858
    85 17 NUC 30 LLDTASALVR A03/A11 A03 L R 1.1070 0.0050 0.0002 1859
    80 16 POL 752 LLGCAANWILR A03/A11 A03 L R 1860
    95 19 NUC 44 LLSFLPSDF A03/A11 A03 L F 1861
    95 19 NUC 44 LLSFLPSDFF A03/A11 A03 L F 1862
    95 19 ENV 175 LLVLQAGFF A03/A11 A03 L F 20.0121 0.0006 0.0002 1863
    100 20 ENV 349 LLYPFVQWF A03/A11 A03 L F 1864
    95 19 NUC 45 LSFLPSDFF A03/A11 A03 S F 20.0123 0.0006 0.0002 1865
    95 19 POL 426 LSLDVSAAF A03/A11 A03 S F 1866
    75 15 POL 564 LSLGIHLNPNK A03/A11 A03 S K 1867
    95 19 X 53 LSLRGLPVCA A03/A11 A03 S A 1868
    95 19 POL 521 LSPFLLAQF A03/A11 A03 S F 1869
    95 19 NUC 169 LSTLPETTVVR A03/A11 A03 S R 26.0537 −0.0009 0.0008 1870
    75 15 ENV 16 LSVPNPLGF A03/A11 A03 S F 1871
    100 20 POL 423 LSWLSLDVSA A03/A11 A03 S A 20.0260 0.0048 0.0035 1872
    75 15 POL 3 LSYQHFRK A03/A11 A03 S K 1873
    85 17 POL 99 LTVNEKRR A03/A11 A03 T R 26.0013 −0.0002 −0.0002 1874
    90 18 NUC 119 LVSFGVWIR A03/A11 A03 V R 1090.08 * 0.0028 0.0120 1875
    100 20 POL 377 LVVDFSQFSR A03/A11 A03 V R 1069.20 * 0.0016 0.3600 1876
    95 19 ENV 249 MCLRRFIIF A03/A11 A03 C F 1877
    90 18 POL 550 MDDVVLGAK A03/A11 A03 D K 1878
    90 18 NUC 30 MDIDPYKEF A03/A11 A03 D F 1879
    85 17 ENV 360 MMWYWGPSLY  A01/A03/ A03 M Y 1039.01 * 0.0500 0.0008 1880
    75 15 X 103 MSTTDLEAYF A03/A11 A03 S F 1881
    75 15 X 103 MSTTDLEAYFK A03/A11 A03 S K 1882
    95 19 POL 572 NFLLSLGIH A03/A11 A03 F H 1883
    90 18 NUC 75 NLEDPASR A03/A11 A03 L R 26.0014 −0.0002 −0.0002 1884
    95 19 POL 45 NLNVSIPWTH A03/A11 A03 L H 1885
    95 19 POL 45 NLNVSIPWTHK A03/A11 A03 L K 26.0538 −0.0009 0.0005 1886
    75 15 ENV 15 NLSVPNPLGF A03/A11 A03 L F 1887
    75 15 ENV 215 NSQSPTSNH A03/A11 A03 S H 1888
    90 18 POL 738 NSVVLSRK A03/A11 A03 S K 26.0015 0.0006 0.0010 1889
    100 20 POL 47 NVSIPWTHK A03/A11 A03 V K 1069.18 * 0.0820 0.0570 1890
    90 18 POL 775 PADDPSRGR A03/A11 A03 A R 1150.35 0.0008 0.0002 1891
    80 16 X 11 PARDVLCLR A03/A11 A03 A R 1150.36 0.0002 0.0002 1892
    90 18 POL 366 PARVTGGVF A03/A11 A03 A F 1893
    75 15 ENV 83 PASTNRQSGR A03/A11 A03 A R 1894
    85 17 X 68 PCALRFTSAR A03/A11 A03 C R 1895
    90 18 ENV 26 PDHQLDPAF A03/A11 A03 D F 1896
    95 19 POL 523 PFLLAQFTSA A03/A11 A03 F A 1897
    95 19 POL 645 PFTQCGYPA A03/A11 A03 F A 1898
    100 20 ENV 244 PGYRWMCLR A03/A11 A03 G R 1.0964 0.0008 0.0005 1899
    95 19 ENV 244 PGYRWMCLPR A03/A11 A03 G R 1.1068 0.0048 0.0001 1900
    90 18 POL 616 PIDWKVCQR A03/A11 A03 I R 1.0985 0.0002 0.0005 1901
    100 20 ENV 391 PIFFCLWVY A03/A11 A03 I Y 1.0843 0.0011 0.0002 1902
    80 16 POL 496 PIILGFRK A03/A11 A03 I K 1903
    95 19 POL 20 PLEEELPR A03/A11 A03 L R 26.0016 0.0002 −0.0002 1904
    100 20 POL 438 PLHPAAMPH A03/A11 A03 L H 20.0128 0.0012 0.0002 1905
    95 19 ENV 17 PLLVLQAGF A03/A11 A03 L F 1906
    95 19 ENV 174 PLLVLQAGFF A03/A11 A03 L F 1907
    100 20 POL 2 PLSYQHFR A03/A11 A03 L R 26.0017 −0.0002 −0.0002 1908
    75 15 POL 2 PLSYQHFRK A03/A11 A03 L K 1.0161 0.0011 0.0031 1909
    85 17 POL 98 PLTVNEKR A03/A11 A03 L R 26.0018 0.0002 −0.0002 1910
    85 17 POL 98 PLTVNEKRR A03/A11 A03 L R 1.0974 0.0008 0.0005 1911
    80 16 POL 516 PMGVGLSPF A03/A11 A03 M F 1912
    80 16 POL 516 PMGVGLSPF A03/A24 A03 M F 1913
    90 18 X 20 PVGAESRGR A03/A11 A03 V R 1.0990 0.0002 0.0005 1914
    85 17 POL 612 PVNRPIDWK A03/A11 A03 V K 1142.06 * 0.0310 0.1400 1915
    95 19 POL 865 QAFTFSPTY A03/A11 A03 A Y 20.0127 0.0030 0.0017 1916
    95 19 POL 854 QAFTFSPTYK A03/A11 A03 A K 1090.10 * 0.0450 0.5400 1917
    80 16 ENV 179 QAGFRLLTR A03/A11 A03 A R 1918
    80 16 ENV 118 QAMQWNSTTF A03/A11 A03 A F 1919
    75 15 NUC 189 QSPRRRRSQSR A03/AAA A03 S R 28.0839 1920
    80 16 POL 189 QSSGILSR A03/A11 A03 S R 1921
    95 19 POL 539 RAFPHCLAF A03/A11 A03 A F 20.0125 0.0015 0.0007 1922
    75 15 POL 106 RLKLIMPAR A03/A11 A03 L R 1.0975 * 0.0950 0.0002 1923
    75 15 POL 106 RLKLIMPARF A03/A11 A03 L F 1924
    75 15 X 128 ALKVFVLGGCR A03/A11 A03 L R 1925
    95 19 POL 387 ALVVDFSCF A03/A11 A03 L F 20.0122 0.0006 0.0002 1926
    95 19 POL 376 RLVVDFSQFSR A03/A11 A03 L R 26.0539 * 0.2800 3.8000 1927
    95 19 NUC 183 RSPRRRTPSPR A03/A11 A03 S R 26.0540 −0.0007 −0.0003 1928
    75 15 NUC 167 RSQSPRRR A03/A11 A03 S R 1929
    75 15 NUC 167 RSQSPRRRR A03/A11 A03 S R 1930
    95 19 NUC 188 RTPSPRRR A03/A11 A03 T R 26.0019 −0.0002 −0.0002 1931
    95 19 NUC 188 RTPSPRRRR A03/A11 A03 T R 1.0971 * 0.0054 0.0005 1932
    80 16 POL 829 RVHFASPLH A03/A11 A03 V H 1933
    100 20 POL 357 RVTGGVFLVDK A03/A11 A03 V K 1147.18 * 0.0190 0.0290 1934
    90 18 X 65 SAGPCALR A03/A11 A03 A R 26.0020 −0.0002 0.0020 1935
    90 18 X 65 SAGPCALRF A03/A11 A03 A F 26.0152 −0.0003 0.0004 1936
    95 19 POL 520 SAICSVVR A03/A11 A03 A R 26.0021 −0.0002 0.0071 1937
    95 19 POL 520 SAICSVVRR A03/A11 A03 A R 1090.11 * 0.0058 0.2100 1938
    90 18 POL 771 SALNPADDPSR A03/A11 A03 A R 26.0542 −0.0004 −0.0003 1939
    100 20 POL 165 SASFCGSPY  A01/A03/ A03 A Y * 1940
    75 15 POL 759 SFPWLLGCA A03/A11 A03 F A 1941
    75 15 POL 759 SFPWLLGCAA A03/A11 A03 F A 1942
    95 19 POL 427 SLDVSAAFYH A03/A11 A03 L H 1943
    75 15 POL 565 SLGIHLNPNK A03/A11 A03 L K 28.0758 * 1944
    100 20 ENV 348 SLLVPFVQWF A03/A11 A03 L F 1945
    95 19 X 54 SLRGLPVCAF A03/A11 A03 L F 20.0259 0.0004 0.0002 1946
    90 18 X 64 SSAGPCALR A03/A11 A03 S F 26.0153 * 0.0080 0.1400 1947
    90 18 X 64 SSAGPCALRF A03/A11 A03 S F 26.0374 −0.0003 −0.0002 1948
    95 19 NUC 170 STLPETTVVR A03/A11 A03 T R 1069.21 * 0.0007 0.0600 1949
    95 19 NUC 170 STLPETTVVRR A03/A11 A03 T R 1083.01 0.0150 1.4000 1950
    80 16 ENV 85 STNRQSGR A03/A11 A03 T R 1951
    75 15 X 104 STTDLEAYF A03/A11 A03 T F 1952
    75 15 X 104 STTDLEAYFK A03/A11 A03 T K 1.0584 * 0.0066 2.7000 1953
    95 19 POL 535 SVVRRAFPH A03/A11 A03 V H 20.0131 * 0.1100 0.6100 1954
    85 17 POL 727 TAELLAACF A03/A11 A03 A F 1955
    85 17 POL 716 TAELLAACFAR A03/A11 A03 A R 28.0544 0.0006 0.0023 1956
    90 18 POL 747 TDNSVVLSR A03/A11 A03 D R 1957
    90 18 POL 747 TDNSVVLSRK A03/A11 A03 D K 20.0264 0.0006 0.0017 1958
    75 15 NUC 138 TFGREIVLEY A03/A11 A03 F Y 1959
    95 19 POL 668 TFSPTYKAF A03/A24 A03 F F 5.0064 1960
    100 20 POL 370 TGGVFLVDK A03/A11 A03 G K 20.0133 0.0007 0.0061 1961
    95 19 NUC 171 TLPETTVVR A03/A11 A03 L R 1.0969 0.0008 0.0002 1962
    95 19 NUC 171 TLPETTVVRR A03/A11 A03 L R 1069.22 * 0.0007 0.0230 1963
    95 19 NUC 171 TLPETTVVRRR A03/A11 A03 L R 26.0545 * 0.0005 0.0160 1964
    100 20 POL 150 TLWKAGILY A03/A11 A03 L Y 1099.03 * 0.1300 0.0008 1965
    100 20 POL 150 TLWKAGILYK A03/A11 A03 L K 1069.15 * 5.3000 0.3800 1966
    100 20 POL 150 TLWKAGILYKR A03/A11 A03 L R 26.0546 0.0082 0.0095 1967
    95 19 POL 519 TSAICSVVR A03/A11 A03 S R 5.0057 0.0005 0.0008 1968
    95 19 POL 519 TSAICSVVRR A03/A11 A03 S R 1142.08 * 0.0018 0.0006 1969
    75 15 POL 758 TSFPWLLGCA A03/A11 A03 S A 1970
    80 16 POL 775 TSFVYVPSA A03/A11 A03 S A 1971
    75 15 X 105 TTDLEAYFK A03/A11 A03 T K 1.0215 * 0.0006 0.9200 1972
    75 15 ENV 278 TTSTGPCK A03/A11 A03 T K 1973
    80 16 NUC 175 TTVVRRRGR A03/A11 A03 T R 1.0970 0.0008 0.0005 1974
    80 16 NUC 176 TVVRRRGR A03/A11 A03 V R 3.0324 0.0003 0.0001 1975
    80 16 NUC 176 TVVRRRGRSPR A03/A11 A03 V R 28.0837 1976
    100 20 POL 373 VFLVDKNPH A03/A11 A03 F H 1977
    80 16 X 131 VFVLGGCRH A03/A11 A03 F H 1978
    75 15 X 131 VFVLGGCRH A03/A11 A03 F H 1979
    95 19 POL 637 VGLLGFAAPF A03/A11 A03 G F 1980
    85 17 POL 96 VGPLTVNEK A03/A11 A03 G K 20.0132 0.0007 0.0078 1981
    85 17 POL 96 VGPLTVNEKR A03/A11 A03 G R 1982
    95 19 POL 554 VLGAKSVQH A03/A11 A03 L H 1983
    90 18 X 133 VLGGCRHK A03/A11 A03 L K 26.0022 0.0150 0.0002 1984
    80 16 ENV 177 VLQAGFFLLTR A03/A11 A03 L R 1985
    85 17 POL 752 VLSRKYTSF A03/A11 A03 S R 1986
    90 18 NUC 120 VSFGVWIR A03/A11 A03 S R 26.0023 * 0.0040 0.0290 1987
    100 20 POL 48 VSIPWTHK A03/A11 A03 S K 26.0024 * 0.0130 0.0170 1988
    100 20 POL 358 VTGGVFLVDK A03/A11 A03 T K 1069.17 * 0.0390 0.0920 1989
    100 20 POL 378 VVDFSQFSR A03/A11 A03 V R 1069.19 * 0.0015 0.0750 1990
    90 18 POL 553 VVLGAKSVQH A03/A11 A03 V H 1991
    85 17 POL 751 VVLSRKYTSF A03/A11 A03 V F 20.0261 0.0004 0.0002 1992
    80 16 NUC 177 VVRRRGRSPR A03/A11 A03 V R 1.1074 0.0027 0.0001 1993
    80 16 NUC 177 VVRRRGRSPRR A03/A11 A03 V R 28.0838 1994
    90 18 NUC 131 WFHISCLTF A03/A11 A03 F F 13.0073 * 1995
    90 18 NUC 131 WFHISCLTF A03/A24 A03 F F 13.0073 * 1996
    85 17 NUC 28 WGMDIDPYK A03/A11 A03 G K 26.0154 −0.0003 0.0006 1997
    85 17 POL 589 WGYSLNFMGY A03/A11 A03 G Y 1998
    80 16 POL 770 WLRGTSFVY A03/A11 A03 I Y 1.0572 0.0076 0.0011 1999
    95 19 NUC 125 WIRTPPAYR A03/A11 A03 I R 1.0968 0.0008 0.0005 2000
    90 18 POL 314 WLQFRNSK A03/A11 A03 L K 26.0025 −0.0002 0.0005 2001
    95 19 POL 425 WLSLDVSAAF A03/A11 A03 L F 2002
    85 17 NUC 26 WLWGMDIDPY A03/A11 A03 L Y 1.0774 * 0.0002 0.0002 2003
    85 17 NUC 26 WLWGMDIDPYK A03/A11 A03 L K 26.0547 0.0030 0.0013 2004
    95 19 ENV 248 WMCLRRFIIF A03/A11 A03 M F 20.0266 0.0004 0.0011 2005
    95 19 ENV 248 WMCLRRFIIF A03/A24 A03 M F 20.0266 0.0004 0.0011 2006
    100 20 POL 122 YLPLDKGIIK A03/A11 A03 L K 1.0173 0.0001 0.0001 2007
    90 18 NUC 118 YLVSFGVWIR A03/A11 A03 L R 1090.13 * 0.0005 0.0002 2008
    90 18 POL 538 YMDDVVLGAK A03/A11 A03 M K 1090.15 * 0.0330 0.0043 2009
    80 16 POL 504 YSHPIILGF A03/A11 A03 S F 2010
    80 16 POL 493 YSHPIILGFR A03/A11 A03 S R 2011
    80 16 POL 493 YSHPIIGFRK A03/A11 A03 S K 2012
    248
  • TABLE XVII
    HBV A24 Motif With Binding Information
    SEQ ID
    Conservancy Frequency Protein Position Sequence P2 C-term Peptide Filed A*2401 NO:
    95 19 X 62 AFSSAGPCAL F L 5.0118 0.0012 2013
    90 18 POL 535 AFSYMDDVVL F L 13.0130 0.0009 2014
    80 16 ENV 108 AMQWNSTTF M F 2015
    100 20 NUC 131 AYRPPNAPI Y I 1090.02 * 0.0310 2016
    100 20 NUC 131 AYRPPNAPIL Y L 1069.24 * 0.0042 2017
    90 18 NUC 117 EYLVSFGVW Y W 26.0150 2018
    90 18 NUC 117 EYLVSFGVWI Y I 17.0428 * 2019
    80 16 ENV 182 FFLLTRILTI F I 2020
    80 16 ENV 181 GFFLLTRIL F L 2021
    75 15 ENV 170 GFLGPLLVL F L 2022
    85 17 NUC 29 GMDIDPYKEF M F 26.0372 2023
    85 17 ENV 65 GWSPQAQGI W I 20.0134 0.0024 2024
    85 17 ENV 65 GWSPQAQGIL W L 20.0266 0.0003 2025
    95 19 ENV 234 GYRWMCLRRF Y F 1069.25 * 0.0007 2026
    80 16 POL 820 HFASPLHVAW F W 2027
    100 20 ENV 381 IFFCLWVYI F I 5.0058 0.0087 2028
    80 16 ENV 245 IFLFILLLCL F L 2029
    95 19 POL 395 KFAVPNLQSL F L 5.0114 0.0020 2030
    100 20 POL 121 KYLPLDKGI Y I 2031
    85 17 POL 745 KYTSFPWLL Y L 1069.23 * 5.3000 2032
    80 16 ENV 247 LFILLLCLI F I 2033
    80 16 ENV 247 LFILLLCLIF F F 2034
    85 17 NUC 101 LWFHISCLTF W F 26.0373 2035
    80 16 POL 492 LYSHPIILGF Y F 2.0181 * 1.1000 2036
    95 19 POL 561 NFLLSLGIHL F L 5.0115 0.0099 2037
    80 16 POL 758 NWILRGTSF W F 2035
    95 19 POL 634 PFTQCGYPAL F L 5.0116 0.0002 2039
    95 19 ENV 341 PFVQWFVGL F L 5.0059 0.0003 2040
    80 16 POL 505 PMGVGLSPF M F 2041
    80 16 POL 750 PWLLGCAANW W W 2042
    100 20 POL 51 PWTHKVGNF W F 20.0138 * 0.0290 2043
    75 15 ENV 242 RFIIFLFIL F L 2044
    75 15 ENV 242 RF1IFLFILL F L 2045
    95 19 ENV 236 RWMCLRRFI W I 20.0135 * 0.0710 2046
    95 19 ENV 236 RWMCLRRFII W I 20.0269 * 1.1000 2047
    100 20 POL 167 SFCGSPYSW F W 20.0139 * 0.0710 2048
    80 16 POL 765 SFVYVPSAL F L 2049
    100 20 ENV 334 SWLSLLVPF W F 20.0136 * 0.3900 2050
    95 19 POL 392 SWPKFAVPNL W L 20.0271 * 5.6000 2051
    95 19 ENV 197 SWWTSLNFL W L 20.0137 * 0.3800 2052
    75 15 POL 4 SYQHFRKLL Y L 2.0042 0.0051 2053
    75 15 POL 4 SYQHFRKLLL Y L 2.0173 * 0.0680 2054
    95 19 POL 657 TFSPTYKAF F F 5.0064 0.0060 2055
    95 19 POL 657 TFSPTVKAFL F L 5.0117 0.0043 2056
    95 19 POL 686 VFADATPTGW F W 20.0272 * 0.0180 2057
    90 18 NUC 102 WFHISCLTF F F 13.0073 * 0.0300 2058
    95 19 ENV 345 WFVGLSPTVW F W 20.0270 0.0120 2059
    95 19 env 237 WMCLRAFIIF M F 20.0266 0.0013 2060
    48
  • TABLE XVIII
    DR SUPER MOTIF (With binding information)
    SEQ ID
    NO: Sequence Peptide DR1 DR2w2B1 DR2w2B1 DR3 DR4w4 Dr4w15
    2061 AANWILRGTSFVYVP 1298.07 0.0920 0.0240 0.0061 0.0023 0.0510 0.0250
    2062 AEDLNLGNLNVSIPW 1186.01 0.0001 −0.0005 −0.0007
    2063 AELLAACFARSRSGA
    2064 AFSYMDDVVLGAKSV 1186.02 0.0027 −0.0005 0.0130 2.9000
    2065 AGFFLLTRILTIPQS 1280.06 4.6000 0.0420 0.0190 0.0040 5.3000 0.1500
    2066 AGPLEEELPRLADEG 35.0091 0.0022
    2067 AKLIGTDNSVVLSRK
    2068 ANWILRGTSFVYVPS
    2069 ARDVLCLRPVGAESR
    2070 ASALYREALESPEHC
    2071 ASKLCLGWLWGMDID 1186.03 0.0002 −0.0005 0.0017
    2072 CLIFLLVLLDYQGML
    2073 CLTFGRETVLEYLVS
    2074 CPGYRWMCLRRFIIF
    2075 CPTVQASKLCLGWLW
    2076 CQVFADATPTGWGLA
    2077 CSVVRRAFPHCLAFS 1186.04 0.1000 0.1024 0.0770 0.0032 0.0016 −0.0022
    2078 CTCIPIPSSWAFARF
    2079 CWWLQFRNSKPCSDY
    2080 DDVVLGAKSVQHLES
    2081 DEGLNRRVAEDLNLG
    2082 DLNLGNLNVSIPWTH 1280.07 0.0038 0.0240
    2083 DVVLGAKSVQHLESL
    2084 DWKVCQRIVGLLGFA 1186.05 0.0120 −0.0026 0.0030
    2085 EIRLKVFVLGGCRHK
    2086 ESRLVVDFSQFSRGN 35.0096 0.0007 2.6000
    2087 FFLLTRILTIPQSLD F064.01
    2088 FGVWIRTPPAYRPPN
    2089 FIIFLFILLLCLIFL
    2090 FLFILLLCLIFLLVL
    2091 FPWLLGCAANWILRG
    2092 FRKLPVNRPIDWKVC
    2093 FSWLSLLVPFVQWFV
    2094 FSYMDDVVLGAKSVQ
    2095 FVQWFVGLSPTVWLS 1186.06 0.4700 0.0035 0.0610 −0.0013 0.0130
    2096 GAHLSLRGLPVCAFS 1186.07 0.7800 0.0042 −0.0041 0.0011
    2097 GFFLLTRILTIPQSL 1280.08 0.4300 0.0150 0.0110 3.1000 0.4500
    2098 GIHLNPNKTKRWGYS
    2099 GLPVCAFSSAGPCAL
    2100 GLYFPAGGSSSGTVN
    2101 GTNLSVPNPLGFFPD
    2102 GTSFVYVPSALNPAD 1280.09 0.3500 0.0140 0.0500 −0.0006 0.3800 0.4100
    2103 GVFLVDKNPHNTTES
    2104 GVGLSPFLLAQFTSA
    2105 GVWIRTPPAYRPPNA 27.0280 0.3700 0.0420 7.2000 0.0120 3.4000 0.5700
    2106 HGGLLGWSPQAQGIL
    2107 HLPLHPAAMPHLLVG
    2108 HLSLRGLPVCAFSSA 1280.10 1.3000 0.0028
    2109 HTALRQAILCWGELM
    2110 HTLWKAGILYKRETT
    2111 IFLFILLLCLIFLLV 1280.11 0.0005 0.0041
    2112 IIFLFILLLCLIFLL 1280.12
    2113 ILGFRKIPMGVGSLP
    2114 ILLLCLIFLLVLLDY F107.01 0.0026 0.0069 0.0320
    2115 IRDLLDTASALYREA
    2116 IRQLLWFHISCLTFG
    2117 IVGLLGFAAPFTQCG 1186.09 0.0200 −0.0005 −0.0007
    2118 IWMMWYWGPSLYNIL
    2119 KFAVPNLQSLTNLLS 1280.13 0.0180 0.0005 −0.0003 0.1300
    2120 KIPMGVGLSPFLLAQ
    2121 KLHLYSHPIILGFRK
    2122 KQAFTFSPTYKAFLC 1298.06 0.5300 0.2400 0.1400 0.0090 1.1000 0.2200
    2123 KQCFRKLPVNRPIDW 1298.04 1.5000 0.0022 0.0210 −0.0006 1.2000 0.8500
    2124 KRRLKLIMPARFYPN
    2125 LAQFTSAICSVVRRA 1186.10 0.0120 0.0065 0.1500 −0.0009 0.0150 0.0280
    2126 LCLIFLLVLLDYQGM F107.02 0.0016 0.0060 0.0230
    2127 LCQVFADATPTGWGL 1280.14 0.0020 0.9600
    2128 LEYLVSFGVWIRTPP
    2129 LFILLLCLIFLLVLL
    2130 LGFFPDHQLDPAFGA
    2131 LGNLNVSIPWTHKVG
    2132 LGPLLVLQAGFFLLT
    2133 LGWLWGMDIDPYKEF 1186.12 0.0004 0.0006 0.0200 0.0280
    2134 LHLYSHPIILGFRKI 1280.16 0.0220 0.0340 0.0400 0.0040 0.6800
    2135 LHTLWKAGILYKRET
    2136 LKVFVLGGCRHKLVC
    2137 LLCLIFLLVLLDYQG 1280.16
    2138 LLDYQGMLPVCPLIP
    2139 LLGFAAPFTQCGYPA
    2140 LLWFHISCLTFGRET
    2141 LPKVLHKRTLGLSAM
    2142 LPLLPIFFCLWVYIZ
    2143 LQSLTNLLSSNLSWL F107.03 2.5000 0.4400 0.0200 −0.0013 4.8000
    2144 LSAMSTTDLEAYFKD
    2145 LSTLPETTVVRRRGR
    2146 LSWLSLDVSAAFYHI
    2147 LTNLLSSNLSWLSLD 1186.14 0.0010 0.0083 0.0160
    2148 LVLLDYQGMLPVCPL 1280.17 0.0034 −0.0013
    2149 LVPFVQWFVGLSPTV 1186.15 0.0130 0.6900 0.0140 −0.0013 0.1500
    2150 MQLFHLCLIISCSCP
    2151 NAPILSTLPETTVVR 1186.16 0.0009 0.0009 −0.0007
    2152 NLNVSIPWTHKVGNF 1186.17 0.0001 −0.0005 −0.0041 −0.0007
    2153 NLSWLSLDVSAAFYH 1186.18 0.1400 0.0003 −0.0005 1.3000 0.2900
    2154 NRPIDWKVCQRIVGL
    2155 PAAMPHLLVGSSGLS
    2156 PDRVHFASPLHVAWR 1298.08 0.0510 0.0290 0.0008 0.0008
    2157 PFLLAQFTSAICSVV F107.04 0.1800 0.0270 0.0042 −0.0013 0.0800
    2158 PHCLAFSYMDDVVLG
    2159 PIILGFRKIPMGVGL
    2160 PLPIHTAELLAACFA 1280.18 0.0046 0.0490
    2161 PPAYRPPNAPILSTL 1186.20 0.0056 −0.0005 0.0038
    2162 PQAMQWNSTTFHQTL 1298.01 0.0012 0.0300
    2163 PQSLDSWWTSLNFLG
    2164 QCGYPALMPLYACIQ 1186.21 0.0062 0.0018 0.0068
    2165 QLLWFHISCLTFGRE
    2166 QQYVGPLTVNEKRRL
    2167 QWFVGLSPTVWLSVI
    2168 RDLLDTASALYREAL 1280.19 0.0001 0.0092
    2169 RDVLCLRPVGAESRG
    2170 RFIIFLFILLLCLIF
    2171 RFSWLSLLVPFVQWF 1186.22 0.0430 0.0008 −0.0007
    2172 RPGLCQVFADATPTG
    2173 RQLLWFHISCLTFGR 1186.23 0.0002 0.0009 0.0140
    2174 RRAFPHCLAFSYMDD F107.05 0.0010 0.0010 −0.0009
    2175 RRFIIFLFILLLCLI
    2176 RRSFGVEPSGSGHID
    2177 RVSWPKFAVPNLQSL
    2178 RWGYSLNFMGYVIGS
    2179 SFGVWIRTPPAYRPP 1186.25 0.0094 0.0110 0.4300 −0.0009 0.0780 0.0630
    2180 SFPWLLGCAANWILR
    2181 SFVYVPSALNPADDP
    2182 SGFLGPLLVLQAGFF
    2183 SPFLLAQFTSAICSV 1186.26 0.1200 0.0200 0.0085 −0.0013 0.0740 0.0190
    2184 SSNLSWLSLDVSAAF 1186.27 0.1400 0.0030 −0.005 1.5000 0.2700
    2185 SVELLSFLPSDFFPS
    2186 SVRFSWLSLLVPFVQ 1280.20 0.9000 0.0099
    2187 SVVLSRKYTSFPWLL 27.0282 0.0005 0.0057 0.2100 −0.0016
    2188 TNFLLSLGIHLNPNK 1298.03 3.5000 0.0410 0.1200 0.0220 0.0360
    2189 TNLLSSNLSWLSLDV 1186.28 0.0016 −0.0005 0.1300
    2190 TRILTIPQSLDSWWT
    2191 TSFVYVPSALNPADD
    2192 TSGFLGPLLVLQAGF
    2193 VAPLPIHTAELLAAC
    2194 VCAFSSAGPCALRFT 1186.29 0.2100 0.2600 0.0023
    2195 VELLSFLPSDFFPSI
    2196 VGLLGFAAPFTQCGY 1280.21 0.0470 0.3100 0.008 −0.0014
    2197 VGNFTGLYSSTVPVF 1298.02 1.7000 0.0100 0.0016 0.0140 0.1700
    2198 VLCLRPVGAESRGRP
    2199 VQWFVGLSPTVWLSV
    2200 WASVRFSWLSLLVPF
    2201 WLSLDVSAAFYHIPL
    2202 WLSLLVPFVQWFVGL
    2203 WMCLRRFIIFLFILL
    2204 WPKFAVPNLQSLTNL 1186.30 0.0007 0.0013 0.0023
    2205 YPALMPLYACIQSKQ 1298.05 0.2400 0.0014
    SEQ ID
    NO: Sequence DR5w11 DR5w12 DR6w19 DR7 DR8w2 DR9 DRW53
    2061 AANWILRGTSFVYVP 0.0140 0.3700 0.0250 0.5800 0.2500 0.2700
    2062 AEDLNLGNLNVSIPW −0.0002 −0.0003 0.0170
    2063 AELLAACFARSRSGA
    2064 AFSYMDDVVLGAKSV 0.0006 −0.0003 −0.0005
    2065 AGFFLLTRILTIPQS 3.6000 0.0700 0.3700 3.1000 0.2600 1.3000
    2066 AGPLEEELPRLADEG
    2067 AKLIGTDNSVVLSRK
    2068 ANWILRGTSFVYVPS
    2069 ARDVLCLRPVGAESR
    2070 ASALYREALESPEHC
    2071 ASKLCLGWLWGMDID −0.0002 0.0013 0.0010
    2072 CLIFLLVLLDYQGML
    2073 CLTFGRETVLEYLVS
    2074 CPGYRWMCLRRFIIF
    2075 CPTVQASKLCLGWLW
    2076 CQVFADATPTGWGLA
    2077 CSVVRRAFPHCLAFS 0.0008 −0.0013 0.0540 0.0590 0.0250 1.2000 0.0460
    2078 CTCIPIPSSWAFARF
    2079 CWWLQFRNSKPCSDY
    2080 DDVVLGAKSVQHLES
    2081 DEGLNRRVAEDLNLG
    2082 DLNLGNLNVSIPWTH 0.0010
    2083 DVVLGAKSVQHLESL
    2084 DWKVCQRIVGLLGFA 0.2500 0.0018 0.0130
    2085 EIRLKVFVLGGCRHK
    2086 ESRLVVDFSQFSRGN
    2087 FFLLTRILTIPQSLD
    2088 FGVWIRTPPAYRPPN
    2089 FIIFLFILLLCLIFL
    2090 FLFILLLCLIFLLVL
    2091 FPWLLGCAANWILRG
    2092 FRKLPVNRPIDWKVC
    2093 FSWLSLLVPFVQWFV
    2094 FSYMDDVVLGAKSVQ
    2095 FVQWFVGLSPTVWLS 0.0072 0.0021 0.0190 0.0690 0.0180 0.0410 0.0044
    2096 GAHLSLRGLPVCAFS 0.0025 0.0077 0.0150
    2097 GFFLLTRILTIPQSL 2.3000 0.0780 3.5000 1.6000 0.5500
    2098 GIHLNPNKTKRWGYS
    2099 GLPVCAFSSAGPCAL
    2100 GLYFPAGGSSSGTVN
    2101 GTNLSVPNPLGFFPD
    2102 GTSFVYVPSALNPAD 0.0470 −0.0001 0.0001 0.2700 0.0610 0.3400
    2103 GVFLVDKNPHNTTES
    2104 GVGLSPFLLAQFTSA
    2105 GVWIRTPPAYRPPNA 0.4800 0.0140 −0.0004 0.2200 0.5300 0.0450
    2106 HGGLLGWSPQAQGIL
    2107 HLPLHPAAMPHLLVG
    2108 HLSLRGLPVCAFSSA 0.0130
    2109 HTALRQAILCWGELM
    2110 HTLWKAGILYKRETT
    2111 IFLFILLLCLIFLLV 0.0018
    2112 IIFLFILLLCLIFLL
    2113 ILGFRKIPMGVGSLP
    2114 ILLLCLIFLLVLLDY 0.0018 0.0047
    2115 IRDLLDTASALYREA
    2116 IRQLLWFHISCLTFG
    2117 IVGLLGFAAPFTQCG −0.0002 0.0009 0.0087
    2118 IWMMWYWGPSLYNIL
    2119 KFAVPNLQSLTNLLS 0.0043 0.0088 −0.0003 0.0056
    2120 KIPMGVGLSPFLLAQ
    2121 KLHLYSHPIILGFRK
    2122 KQAFTFSPTYKAFLC 0.2400 0.0024 0.0200 0.3300 0.1200 0.5400
    2123 KQCFRKLPVNRPIDW 0.0130 0.0013 0.0043 0.4000 0.0580 0.0250
    2124 KRRLKLIMPARFYPN
    2125 LAQFTSAICSVVRRA 0.0076 0.0091 0.0010 0.0280 0.0150 0.0880 0.0190
    2126 LCLIFLLVLLDYQGM 0.0017 0.0044
    2127 LCQVFADATPTGWGL 0.0013
    2128 LEYLVSFGVWIRTPP
    2129 LFILLLCLIFLLVLL
    2130 LGFFPDHQLDPAFGA
    2131 LGNLNVSIPWTHKVG
    2132 LGPLLVLQAGFFLLT
    2133 LGWLWGMDIDPYKEF −0.0002 0.0004 0.0430
    2134 LHLYSHPIILGFRKI 0.0410 0.0310 0.0002 0.0006 0.0610 0.0490
    2135 LHTLWKAGILYKRET
    2136 LKVFVLGGCRHKLVC
    2137 LLCLIFLLVLLDYQG
    2138 LLDYQGMLPVCPLIP
    2139 LLGFAAPFTQCGYPA
    2140 LLWFHISCLTFGRET
    2141 LPKVLHKRTLGLSAM
    2142 LPLLPIFFCLWVYIZ
    2143 LQSLTNLLSSNLSWL 0.0680 0.7500 0.0260 0.1500 0.0880 0.1100
    2144 LSAMSTTDLEAYFKD
    2145 LSTLPETTVVRRRGR
    2146 LSWLSLDVSAAFYHI
    2147 LTNLLSSNLSWLSLD 0.0013 0.0019 0.0200
    2148 LVLLDYQGMLPVCPL 0.0011
    2149 LVPFVQWFVGLSPTV 0.3800 0.6600 0.0018 0.0092 0.6600 2.5000 2.6000
    2150 MQLFHLCLIISCSCP
    2151 NAPILSTLPETTVVR −0.0002 0.0005 0.1600
    2152 NLNVSIPWTHKVGNF −0.0002 0.0005 0.0009
    2153 NLSWLSLDVSAAFYH 0.0033 0.0022 0.0330 0.0041 0.0150 0.0820 2.4000
    2154 NRPIDWKVCQRIVGL
    2155 PAAMPHLLVGSSGLS
    2156 PDRVHFASPLHVAWR 0.0008 0.0190 0.0810 0.0035 0.2400
    2157 PFLLAQFTSAICSVV 0.0120 0.0016 0.0800 0.0770 0.0680 0.0590
    2158 PHCLAFSYMDDVVLG
    2159 PIILGFRKIPMGVGL
    2160 PLPIHTAELLAACFA −0.0003
    2161 PPAYRPPNAPILSTL 0.0022 0.0024 0.0015
    2162 PQAMQWNSTTFHQTL 0.1200
    2163 PQSLDSWWTSLNFLG
    2164 QCGYPALMPLYACIQ 0.0023 0.0006
    2165 QLLWFHISCLTFGRE
    2166 QQYVGPLTVNEKRRL
    2167 QWFVGLSPTVWLSVI
    2168 RDLLDTASALYREAL 0.0770
    2169 RDVLCLRPVGAESRG
    2170 RFIIFLFILLLCLIF
    2171 RFSWLSLLVPFVQWF 0.0002 0.0005 0.0031
    2172 RPGLCQVFADATPTG
    2173 RQLLWFHISCLTFGR 0.0011 0.0061 0.0098
    2174 RRAFPHCLAFSYMDD 0.0010 0.0017
    2175 RRFIIFLFILLLCLI
    2176 RRSFGVEPSGSGHID
    2177 RVSWPKFAVPNLQSL
    2178 RWGYSLNFMGYVIGS
    2179 SFGVWIRTPPAYRPP 0.0260 0.0071 0.0002 0.0240 0.2500 0.2800 0.0016
    2180 SFPWLLGCAANWILR
    2181 SFVYVPSALNPADDP
    2182 SGFLGPLLVLQAGFF
    2183 SPFLLAQFTSAICSV −0.0002 −0.0013 0.0540 0.0330 0.0014 0.0380 0.2000
    2184 SSNLSWLSLDVSAAF 0.0046 0.0180 0.1000 0.0038 0.0460 0.0110 6.2000
    2185 SVELLSFLPSDFFPS
    2186 SVRFSWLSLLVPFVQ 0.0037
    2187 SVVLSRKYTSFPWLL 0.5300 0.0130
    2188 TNFLLSLGIHLNPNK 0.0053 0.0160 0.2200 0.0032 0.3800
    2189 TNLLSSNLSWLSLDV 0.0006 0.0019 0.0410
    2190 TRILTIPQSLDSWWT
    2191 TSFVYVPSALNPADD
    2192 TSGFLGPLLVLQAGF
    2193 VAPLPIHTAELLAAC
    2194 VCAFSSAGPCALRFT 0.0003 0.0200 0.0150
    2195 VELLSFLPSDFFPSI
    2196 VGLLGFAAPFTQCGY −0.0004 −0.0001 0.0014 0.5700
    2197 VGNFTGLYSSTVPVF 0.0035 0.0580 0.5600 0.0044 0.3100
    2198 VLCLRPVGAESRGRP
    2199 VQWFVGLSPTVWLSV
    2200 WASVRFSWLSLLVPF
    2201 WLSLDVSAAFYHIPL
    2202 WLSLLVPFVQWFVGL
    2203 WMCLRRFIIFLFILL
    2204 WPKFAVPNLQSLTNL 0.0002 0.0008 0.0180
    2205 YPALMPLYACIQSKQ 0.0011
    145
  • TABLE XIX
    HBV DR3 MOTIF PEPTIDES WITH BINDING DATA
    Total Core
    Con- Con- Core SEQ Binding
    ser- ser- Core Pro- Posi- Core SEQ ID ID Data Mo-
    vancy Total vancy Freq. tein tion Sequence NO: Sequence NO: Peptide Filed DR3 tif
    90.00 18 90.00 18 POL 535 YMDDWLGA 2228 AFSYMDDVVLGAKSV 2206 1186.02 0.0130 DR3
    55.00 11 95.00 19 POL 655 PSPTYKAFL 2229 AFTFSPTYKAFLCKQ 2207 35.0099 0.0035 DR3
    65.00 13 90.00 18 POL 18 LEEELPRLA 2230 AGPLEEELPRLADEG 2208 35.0091 0.0022 DR3
    65.00 13 80.00 16 POL 731 IGTDNSVVL 2231 AKLIGTDNSVVLSRK 2209 DR3
    85.00 17 85.00 17 NUC 34 LYREALESP 2232 ASALYREALESPEHC 2210 DR3
    70.00 14 75.00 15 NUC 136 RGRETVLEY 2233 CLTFGRETVLEYLVS 2211 DR3
    90.00 18 90.00 18 X 48 AHLSLRGLP 2234 DHGAHLSLRGLPVCA 2212 DR3
    85.00 17 90.00 18 POL 737 VVLSRKYTS 2235 DNSVVLSRKYTSFPW 2213 DR3
    45.00 9 100.00 20 POL 374 LVVDFSQFS 2236 ESRLVVDFSQFSRGN 2214 35.0096 * 2.6000 DR3
    5.00 1 75.00 1 ENV 172 AVLDPRVRG 2237 FHQAVLDPRVRGLYL 2215 DR3
    90.00 18 95.00 19 ENV 256 VLLDYQGML 2238 FLLVLLDYQGMLPVC 2216 35.009 0.0170 DR3
    55.00 11 100.00 20 POL 360 FLVDKNPHN 2239 GGVFLVDKNPHNTTE 2217 35.0095 0.0790 DR3
    95.00 19 95.00 19 POL 683 VFADATPTG 2240 LCQVFADATPTGWGL 2218 1280.14 0.0000 DR3
    35.00 7 95.00 19 X 18 VGAESRGRP 2241 LRPVGAESRGRPVSG 2219 35.0101 −0.0017 DR3
    55.00 11 95.00 19 POL 412 LSLDVSAAF 2242 LSWLSLDVSAAFYHI 2220 DR3
    45.00 9 85.00 17 NUC 27 MDIDPYKEF 2243 LWGMDIDPYKEFGAS 2221 DR3
    85.00 17 100.00 20 POL 34 VAEDLNLGN 2244 NRRVAEDLNLGNLNV 2222 35.0092 0.1400 DR3
    100.00 20 100.00 20 POL 47 IPWTHKVGN 2245 NVSIPWTHKVGNFTG 2223 DR3
    45.00 9 95.00 19 ENV 10 FFPDHQLDP 2246 PLGFFPDHQLDPAFG 2224 DR3
    30.00 6 75.00 15 POL 241 PGVEPSGSG 2247 RRSFGVEPSGSGHID 2225 DR3
    100.00 20 100.00 20 POL 120 LPLDKGIKP 2248 TKYLPLDKGIKPYYP 2226 35.0084 −0.0017 DR3
    60.00 12 85.00 17 POL 96 LTVNEKRRL 2249 VGPCTVNEKRRLKLI 2227 35.0093 * 2.2000 DR3
    22 22
  • TABLE XX
    Population coverage with combined HLA Supertypes
    PHENOTYPIC FREQUENCY
    N.A.
    HLA-SUPERTYPES Caucasian Black Japanese Chinese Hispanic Average
    A2, A3, B7 83.0 86.1 87.5 88.4 86.3 86.2
    A2, A3, B7, A24, B44, A1 99.5 98.1 100.0 99.5 99.4 99.3
    A2, A3, B7, A24, B44, A1, 99.9 99.6 100.0 99.8 99.9 99.8
    B27, B62, B58
  • TABLE XXI
    HBV ANALOGS
    A2 A3 B7 1*
    Fixed A1 Super Super A24 Super Anchor SEQ ID
    AA Sequence Nomen. Motif Motif Motif Motif Motif Fixer Analog NO:
    ALFKDWEEL 2250
    9 ALMPLYACV L2.IV9 N Y N N N 1 A 2251
    ALMPLYASI 2252
    9 ALMPLYAXI N Y N N N A 2253
    10 ALPSDFFPSV N Y N N N No A 2254
    ALPSDFFPSV-NH2 2255
    ALSLIVNLL 2256
    9 AMTFSPTYK N N Y N N A 2257
    ATVELLSFLPSDFFPSV-NH2 2258
    10 CILLLCLIFL N Y N N N No A 2259
    11 CILLLCLIFLL N Y N N N No A 2260
    9 DPFRGRLGL N N N N N A 2261
    9 DPSRGRLGI N N N N Y A 2262
    ELLSFLPSDFFPSV-NH2 2263
    10 FAPSDFFPSV LA2.V10 N Y N N N Rev A 2264
    10 FILLLXLIFL N Y N N N A 2265
    10 FLASDFPPSV N Y N N N No A 2266
    10 FLGLSPTVWV VL2.LV1 N Y N N N 1 A 2267
    10 FLKSDFPPSV N Y N N N No A 2268
    10 FLLAQFTSAV L2.IV10 N Y N N N 1 A 2269
    9 FLLAQFTSV L2.AV9 N Y N N N 1 A 2270
    9 FLLPIFFCL N Y N N N No A 2271
    9 FLLSLGIHV L2.LV9 N Y N N N 1 A 2272
    9 FLLTRILTV L2.IV9 N Y N N N 1 A 2273
    9 FLLTRILYI N Y N N N A 2274
    9 FLLTYILTI N Y N N N A 2275
    10 FLMSDYFPSV N Y N N N No A 2276
    9 FLMSYFPSV N Y N N N No A 2277
    10 FLPADFFPSI L2.SA4 N Y N N N Rev A 2278
    10 FLPADFFPSV N Y N N N No A 2279
    10 FLPDDFFPSA L2.SD4 N Y N N N Rev A 2280
    10 FLPDDFFPSV N Y N N N No A 2281
    10 FLPNDFFPSA L2.SN4 N Y N N N Rev A 2282
    10 FLPNDFFPSV N Y N N N No A 2263
    10 FLPS(X)YFPSV N N N N N A 2284
    10 FLPSAFFPSV N Y N N N No A 2285
    10 FLPSD(X)FPSV N N N N N A 2266
    10 FLPSDAFPSV N Y N N N No A 2287
    10 FLPSDFAPSV N Y N N N No A 2288
    FLPSDFF-NH2 2289
    10 FLPSDFFASV N Y N N N No A 2290
    10 FLPSDFFKSV N Y N N N No A 2291
    8 FLPSDFFP N N N N N A 2292
    FLPSDFFP-NH2 2293
    10 FLPSDFFPAV N Y N N N No A 2294
    10 FLPSDFFPKV N Y N N N No A 2295
    9 FLPSDFFPS N N N N N A 2296
    FLPSDFFPS-NH2 2297
    10 FLPSDFFPSA L2.VA10 N Y N N N Rev A 2298
    10 FLPSDFFPSI L2.V110 N Y N N N Rev A 2299
    FLPSDFFPSV(CONH2) 2300
    FLPSDFFPSV-NH2 2301
    11 FLPSDFFPSVR N N Y N N A 2302
    FLPSDFFPSVR-NH2 2303
    12 FLPSDFFPSVRD N N N N N A 2304
    10 FLPSDFYPSV N Y N N N No A 2305
    11 FLPSDLLPSVR N N Y N N A 2306
    10 FLPSDYPPSV N Y N N N No A 2307
    10 FLPSEFFPSV N Y N N N No A 2308
    9 FLPSYPPSA L2.FY5 N Y N N N Rev3 A 2309
    9 FLPSYFPSV L2.FY5 N Y N N N 3 A 2310
    10 FLPSZFFPSV N Y N N N No A 2311
    10 FLPSZFFPSV N Y N N N No A 2312
    10 FLPVDFFPSI L2.SV4 N Y N N N Rev A 2313
    10 FLPVDFFPSV N Y N N N No A 2314
    FLSKQYLNL 2315
    9 FLYTRILTI N Y N N N A 2316
    8 FMFSPTYK N N Y N N A 2317
    10 FMLLLLCLIFL IM2.L10 N Y N Y N 1 A 2318
    10 FMPSDFFPSV LM2.V1 N Y N N N 1 A 2319
    8 FPAAMPHL N Y N N N A 2320
    9 FPAAMPHLL N N N N Y A 2321
    10 FPAAMPHLLV N N N N Y A 2322
    9 FPALMPLYA N N N N Y A 2223
    10 FPARVTGGVF N N N N Y A 2324
    10 FPCALRFTSA N N N N Y A 2325
    9 FPFCLAFSY N N N N Y A 2326
    10 FPFCLAFSYM N N N N Y A 2327
    9 FPHCLAFAL N N N N Y A 2328
    9 FPHCLAFAY N N N N Y A 2329
    9 FPHCLAFSA N N N N Y A 2330
    9 FPHCLAFSI N N N N Y A 2331
    9 FPHCLAFSL N N N N Y A 2332
    10 FPHCLAFSYI N N N N Y A 2333
    10 FPHXLAFSYM N N N N Y A 2334
    9 FPIPSSWAF N N N N Y A 2335
    9 FPSRGRLGL N N N N Y A 2336
    9 FPVCAFSSA N N N N Y A 2337
    9 FPVCLAFSY N N N N Y A 2338
    10 FQPSDYFPSV N Y N N N Rev A 2339
    8 FVFSPTYK N N Y N N A 2340
    9 FVLGGXRHK N N Y N N A 2341
    9 GLQQVFADV L2.AV9 N Y N N N 1 A 2342
    9 GLLGWSPQV L2.AV9 N Y N N N 1 A 2343
    9 GLWIRTPPV VL2.AV9 N Y N N N 1 A 2344
    9 GLXQVFADA N Y N N N A 2345
    10 GMDNSVVLSR N N Y N N A 2346
    11 GMDNSVVLSRK N N Y N N A 2347
    10 GPCALRFTSI N N N N Y A 2348
    10 GPFALRFTSA N N N N Y A 2349
    10 GPXALRFTSA N N N N Y A 2350
    10 GTFNSVVLSR N N Y N N A 2351
    11 GTFNSVVLSRK N N Y N N A 2352
    10 GVDNSVVLSR N N Y N N A 2353
    11 GVDNSVVLSRK N N Y N N A 2354
    10 GYRWMXLRRF N N N Y N A 2355
    9 HISXLTFGR N N Y N N A 2356
    10 HMLWKAGILY Y N Y N N A 2357
    11 HMLWKAGILYK N N Y N N A 2358
    8 HPAAMPHI N N N N Y A 2359
    9 HPAAMPHLI N N N N Y A 2360
    10 HPAAMPHLLI N N N N Y A 2361
    8 HPFAMPHL N N N N Y A 2362
    9 HPFAMPHLL N N N N Y A 2363
    10 HPFAMPHLLV N N N N Y A 2364
    10 HTLWKAGILK N N Y N N A 2365
    10 HTLWKAGILR N N Y N N A 2366
    10 HVLWKAGILY N N Y N N A 2367
    11 HVLWKAGILYK N N Y N N A 2368
    IIKKSEQFV 2369
    9 ILGLLGFAV VL2.AV9 N Y N N N 1 A 2370
    10 ILLLCLIFLV L2.LV10 N Y N N N 1 A 2371
    9 ILLLXLIFL N Y N N N A 2372
    10 ILLLXLIFLL N Y N N N A 2373
    9 IPFPSSWAF N N N N Y A 2374
    9 IPILSSWAF N N N N Y A 2375
    9 IPIPMSWAF N N N N Y A 2376
    9 IPIPSSWAI N N N N Y A 2377
    9 IPITSSWAF N N N N Y A 2376
    KIKESFRKL 2379
    9 KLFLYSHPI N Y N N N No A 2380
    9 KLHLYSHPV L2.IV9 N Y N N N 1 A 2381
    9 KVGNFTGLK N N Y N N A 2382
    9 KVGNFTGLR N N Y N N A 2383
    9 LLAQFTSAV L2.IV9 N Y N N N 1 A 2384
    10 LLFYQGMLPV N Y N N N No A 2385
    LLGSAANWI 2386
    10 LLGXAANWIL N Y N N N A 2387
    9 LLLXLIFLL N Y N N N A 2388
    10 LLLXLIFLLV N Y N N N A 2389
    10 LLLYQGMLPV N Y N N N No A 2390
    9 LLPFVQWFV VL2.V9 N Y N N N 1 A 2391
    10 LLPIFFXLWV N Y N N N A 2392
    LLSFLPSDFFPSV-NH2 2393
    9 LLSSNLSWV L2.LV9 N Y N N N 1 A 2394
    10 LLVLQAGFFV L2.LV10 N Y N N N 1 A 2395
    LLXLIFLLV N Y N N N A 2396
    10 LMLLDYQGMV VM2.LV N Y N N N 1 A 2397
    10 LMLQAGFFLV VM2.LV N Y N N N 1 A 2398
    9 LMPFVQWNFV VM2.V9 N Y N N N 1 A 2399
    9 LPFCAFSSA N N N N Y A 2400
    8 LPIFFCLI N N N N Y A 2401
    9 LPIFFCLWI N N N N Y A 2402
    9 LPIHTAELI N N N N Y A 2403
    11 LPIHTAELLAI N N N N Y A 2404
    LPSDFFPSV-NH2 2405
    9 LPVCAFSSI N N N N Y A 2406
    9 LPVXAFSSA N N N N Y A 2407
    12 LSFLPSDFFPSV N N N N N A 2408
    LSFLPSDFFPSV-NH2 2409
    10 MMWYWGPSLK N N Y N N A 2410
    10 MMWYWGPSLR N N Y N N A 2411
    9 MMWYWGPSV M2.LV9 N Y N N N 1 A 2412
    8 MPLSYQHI N N N N Y A 2413
    9 NLGNLNVSV L2.IV9 N Y N N N 1 A 2414
    NLNNLNVSI 2415
    9 NMGLKYRQL N Y N Y N No A 2416
    11 NPLGFFPDHQI N N N N Y A 2417
    9 PLLPIFFCV L2.LV9 N Y N N N 1 A 2418
    9 PLLPIFFXL N Y N N N A 2419
    8 PSDFFPSV N N N N N A 2420
    PSDFFPSV-NH2 2421
    10 QLLWFHISXL N Y N N N A 2422
    10 QMFTFSPTYK N N Y N N A 2423
    10 QVFTFSPTYK N N Y N N A 2424
    RIPRTPRSV 2425
    9 RLSWPKFAV VL2.V9 N Y N N N 1 A 2426
    9 RLTGGVFLV VL2.V9 N Y N N N 1 A 2427
    9 RMLTIPQSV IM2.LV9 N Y N N N 1 A 2428
    9 RMTGGVFLV VM2.V9 N Y N N N 1 A 2429
    9 RMYLHTLWK N N Y N N A 2430
    9 RVYLHTLWK N N Y N N A 2431
    9 SAIXSVVRR N N Y N N A 2432
    11 SFLPSDFFPSV N N N N N A 2433
    SFLPSDFFPSV-NH2 2434
    9 SLDSWWTSV L2.LV9 N Y N N N 1 A 2435
    SLNFLGGTTV(NH2) 2436
    9 SMICSVVRR N N Y N N A 2437
    10 SMLPETTVVR N N Y N N A 2438
    11 SMLPETTVVRR N N Y N N A 2439
    10 SMLSPFLPLV IM2.LV1 N Y N N N 1 A 2440
    8 SPFLLAQI N N N N Y A 2441
    11 STLPETYVVRR N N Y N N A 2442
    9 SVICSVVRR N N Y N N A 2443
    10 SVLPETTVVR N N Y N N A 2444
    11 SVLPETTVVRR N N Y N N A 2445
    9 SVNRPIDWK N N Y N N A 2446
    9 SVVRRAFPK N N Y N N A 2447
    9 SVVRRAFPR N N Y N N A 2448
    9 TLWKAGILK N N Y N N A 2449
    9 TLWKAGILR N N Y N N A 2450
    10 TMPETTVVRR N N Y N N A 2451
    9 TMWKAGILY Y N Y N N A 2452
    10 TMWKAGILYK N N Y N N A 2453
    10 TPARVTGGVI N N N N Y A 2454
    10 TPFRVTGGVF N N N N Y A 2455
    10 TSAIXSVVRR N N Y N N A 2458
    TVELLSFLPSDFFPSV-NH2 2457
    10 TVPETTVVRR N N Y N N A 2468
    9 TVWKAGILY N N Y N N A 2459
    10 TVWKAGILYK N N Y N N A 2460
    VELLSFLPSDFFPSV-NH2 2461
    VLEYLVSFGV(NH2) 2462
    VLGGSRHKL 2463
    9 VLLDYQGMV L2.LV9 N Y N N N 1 A 2464
    9 VLQAGFFLV L2.LV9 N Y N N N 1 A 2465
    10 VMGGVFLVDK N N Y N N A 2466
    10 VPFVQWFVGI N N N N Y A 2467
    8 VPSALNPI N N N N Y A 2468
    9 WFFSQFSR N N Y N N A 2469
    10 VVGGVFLVDK N N Y N N A 2470
    9 WLLRGTSFV IL2.V9 N Y N N N 1 A 2471
    10 YLFTLWKAGI N Y N N N No A 2472
    10 YLHTLWKAGV L2.IV10 N Y N N N 1 A 2473
    10 YLLTLWKAGI N Y N N N No A 2474
    9 YLLTRILTI N Y N N N A 2475
    9 YLPSDFFPSV VL2.AV9 N Y N N N 1 A 2476
    10 YLPSDFFPSV N Y N N N No A 2477
    9 YMDDVVLGV M2.AV9 N Y N N N 1 A 2478
    9 VMFDVVLGA N Y N N N No A 2479
    10 YMFDVVLGAK N N Y N N A 2480
    10 YNMGLKFRQL N N N N N A 2481
    8 YPALMPLI N N N N Y A 2482
    9 VPALMPLYI N N N N Y A 2483
    9 YPFLMPLVA N N N N Y A 2484
    12 YSFLPSDFFPSV N N N N N A 2485
    236
  • TABLE XXII
    Discreet substitutions improve the B7 supertype binding capacity and
    degeneracy of peptide ligands.
    Figure US20100068228A1-20100318-C00001
  • TABLE XXIII
    Sets of preferred epitopes restricted by class I and
    class II molecules can be selected for inclusion in an
    HBV-specific vaccine. Table XXIII lists as a matter of
    example one such set of epitopes.
    SEQ ID
    Peptide Sequence Protein restriction NO:
    A) Class I restricted epitopes
    924.07 FLPSDFFPSV core 18 A2 2521
    777.03 FLLTRILTI env 183 A2 2522
    927.15 ALMPLYACI pol 642 A2 2523
    1013.01 WLSLLVPFV env 335 A2 2524
    1090.14 YMDDVVLGA pol 538 A2/A1 2525
    1168.02 GLSRYVARL pol 455 A2 2526
    927.11 FLLSLGIHL pol 562 A2 2527
    1069.10 LLPIFFCLWV env 378 A2 2528
    1069.06 LLVPFVQWFV env 338 A2 2529
    1147.16 HTLWKAGILYK pol 149 A3/A1 2530
    1083.01 STLPETTVVRR core 141 A3 2531
    1069.16 NVSIPWTHK pol 47 A3 2532
    1069.20 LVVDFSQFSR pol 388 A3 2533
    1090.10 QAFTFSPTYK pol 665 A3 2534
    1090.11 SAICSVVRR pol 531 A3 2535
    1142.05 KVGNFTGLY pol 629 A3/A1 2536
    1147.05 FPHCLAFSYM pol 530 B7 2537
    988.05 LPSDFPPSV core 19 B7 2538
    1145.04 IPIPSSWAF env 313 B7 2539
    1147.02 HPAAMPHLL pol 429 B7 2540
    26.0570 YPALMPLYACI pol 640 B7 2541
    1147.04 TPARVTGGVF pol 354 B7 2542
    1.0519 DLLDTASALY core 419 A1 2543
    2.0239 LSLDVSAAFY pol 1000 A1 2544
    1039.06 WMMWYWGPSLY env 359 A1 2545
    20.0269 RWMCLRRFII env 236 A24 2546
    20.0136 SWLSLLVPF env 334 A24 2547
    20.0137 SWWTSLNFL env 197 A24 2548
    13.0129 EYLVSFGVWI core 117 A24 2549
    1090.02 AYRPPNAPI core 131 A24 2550
    13.0073 WFHISCLTF core 102 A24 2551
    20.0271 SWPKFAVPNL pol 392 A24 2552
    1069.23 KYTSFPWLL pol 745 A24 2553
    2.0181 LYSHPIILGF pol 492 A24 2554
    B) Glass II restricted epitopes
    F107.03 LQSLTNLLSSNLSWL pol 412 DR 2555
    supermotif
    1298.06 KQAFTESPTYKAFLC pol 664 2556
    1280.06 AGFFLLTRILTIPQS env 180 2557
    1280.09 GTSFVYVPSALNPAD pol 774 2558
    CF-08 VSFGVWIRTPPAYRPPNAPI core 120 2559
    27.0281 RHYLHTLWKAGILYK pol 145 2560
    1186.15 LVPFVQWFVGLSPTV env 339 2561
    1280.15 LHLYSHPIILGFRKI pol 501 2562
    F107.04 PFLLAQFTSAICSVV pol 523 2563
    1298.04 KQCFRKLPVNRPIDW pol 618 2564
    1298.07 AANWILRGTSFVYVP pol 767 2565
    857.02 PHHTALRQAILCWGELMTLA core 50 2566
    1280.14 LCQVFADATPTGWGL pol 694 DR3 motif 2567
    35.0096 ESRLVVDFSQFSRGN pol 385 2568
    35.0093 VGPLTVNEKRRLKLI pol 96 2569
    1186.27 SSNLSWLSLDVSAAF pol 420 2570
    1186.18 NLSWLSLDVSAAFYH pol 442 2571

Claims (58)

1. A peptide composition of less than 100 amino acid residues comprising a peptide epitope useful for inducing an immune response against hepatitis B virus (HBV) said epitope (a) having an amino acid sequence of about 8 to about 13 amino acid residues that have at least 65% identity with a native amino acid sequence for HBV, and, (b) binding to at least one MHC class I HLA allele with a dissociation constant of less than about 500 nM.
2. The composition of claim 1, further wherein said peptide has at least 77% identity with a native HBV amino acid sequence.
3. The composition of claim 1, further wherein said peptide has 100% identity with a native HBV amino acid sequence.
4. The composition of claim 1, further wherein said peptide is one of those peptides described in Tables VI-XVII or XXI.
5. The composition of claim 4, further wherein said peptide is one of the peptides designated as being from the envelope region of HBV.
6. The composition of claim 4, further wherein said peptide is one of the peptides designated as being from the polymerase region of HBV.
7. The composition of claim 4, further wherein said peptide is one of the peptides designated as being from the protein X region of HBV.
8. The composition of claim 4, further wherein said peptide is one of the peptides designated as being from the nucleocapsid core region of HBV.
9. A composition of less than 100 amino acid residues comprising a peptide epitope useful for inducing an immune response against hepatitis B virus (HBV) said peptide (a) having an amino acid sequence of about 8 to about 13 amino acid residues and (b) bearing one of the HLA motifs set out in Tables I and II.
10. The composition of claim 9, further wherein said peptide is one of those peptides described in Table VI or Table XXI bearing an HLA A1 supermotif.
11. The composition of claim 9, further wherein said peptide is one of those peptides described in Table VII or Table XXI bearing an HLA A2 supermotif.
12. The composition of claim 9, further wherein said peptide is one of those peptides described in Table VIII or Table XXI bearing an HLA A3 supermotif.
13. The composition of claim 9, further wherein said peptide is one of those peptides described in Table IX or Table XXI bearing an HLA A24 supermotif.
14. The composition of claim 9, further wherein said peptide is one of those peptides described in Table X or Table XXI bearing an HLA B7 supermotif.
15. The composition of claim 9, further wherein said peptide is one of those peptides described in Table XI bearing an HLA B27 supermotif.
16. The composition of claim 9, further wherein said peptide is one of those peptides described in Table XII bearing an HLA B44 supermotif.
17. The composition of claim 9, further wherein said peptide is one of those peptides described in Table XIII bearing an HLA B58 supermotif.
18. The composition of claim 9, further wherein said peptide is one of those peptides described in Table XIV bearing an HLA B62 supermotif.
19. The composition of claim 9, further wherein said peptide is one of those peptides described in Table XV bearing an HLA A1 motif.
20. The composition of claim 9, further wherein said peptide is one of those peptides described in Table XVI bearing an HLA A3 motif.
21. The composition of claim 9, further wherein said peptide is one of those peptides described in Table XVI bearing an HLA A11 motif.
22. The composition of claim 9, further wherein said peptide is one of those peptides described in Table XVII bearing an HLA A24 motif.
23. The composition of claim 9, further wherein said peptide is one of those peptides described in Table VII bearing an HLA A2.1 motif wherein the epitope is numbered from an amino terminal to carboxyl terminal orientation relative to the peptide, with the proviso that the peptide does not bear L or M at position 2 and V at C-terminal position 9 of a 9 amino acid peptide.
24. An analog of an HBV peptide of less than 100 amino acid residues in length that bears an HLA binding motif, the analog bearing the same HLA binding motif as the peptide but comprising at least one anchor residue that is different from that of the peptide.
25. The composition of claim 24, further wherein said peptide is an analog of a peptide described in Table VI bearing an HLA A1 supermotif.
26. The composition of claim 24, further wherein said peptide is an analog of a peptide described in Table VII bearing an HLA A2 supermotif.
27. The composition of claim 24, further wherein said peptide is an analog of a peptide described in Table VIII bearing an HLA A3 supermotif.
28. The composition of claim 24, further wherein said peptide is an analog of a peptide described in Table IX bearing an HLA A24 supermotif.
29. The composition of claim 24, further wherein said peptide is an analog of a peptide described in Table X bearing an HLA B7 supermotif.
30. The composition of claim 24, further wherein said peptide is an analog of a peptide described in Table XI bearing an HLA B27 supermotif.
31. The composition of claim 24, further wherein said peptide is an analog of a peptide described in Table XII bearing an HLA B44 supermotif.
32. The composition of claim 24, further wherein said peptide is an analog of a peptide described in Table XIII bearing an HLA B58 supermotif
33. The composition of claim 24, further wherein said peptide is an analog of a peptide described in Table XIV bearing an HLA B62 supermotif.
34. The composition of claim 24, further wherein said peptide is an analog of a peptide described in Table XV bearing an HLA A1 motif.
35. The composition of claim 24, further wherein said peptide is an analog of a peptide described in Table XVI bearing an HLA A3 motif.
36. The composition of claim 24, further wherein said peptide is an analog of a peptide described in Table XVI bearing an HLA A11 motif.
37. The composition of claim 24, further wherein said peptide is an analog of a peptide described in Table XVII bearing an HLA A24 motif.
38. The composition of claim 24, further wherein said peptide is an analog of a peptide described in Table VII bearing an HLA A2.1 motif.
39. The composition of claim 24, wherein said peptide is an analog of a peptide described in Table XVIII or Table XIX comprising at HLA class II motif.
40. A composition of less than 100 amino acid residues comprising a peptide epitope useful for inducing an immune response against hepatitis B virus (HBV) said peptide (a) having an amino acid sequence of about 9 to about 25 amino acid residues that have at least 65% identity with a native amino acid sequence for HBV and (b) binding to at least one MHC class II HLA allele with a dissociation constant of less than about 1000 nM.
41. The composition of claim 40, further wherein said peptide has at least 77% identity with a native HBV amino acid sequence.
42. The composition of claim 40, further wherein said peptide has 100% identity with a native HBV amino acid sequence.
43. The composition of claim 40, further wherein said peptide is one of those peptides described in Table XVIII or Table XIX.
44. A peptide composition of less than 100 amino acid residues, said composition comprising an epitope useful for inducing an immune response against hepatitis B virus (HBV) said epitope (a) having an amino acid sequence of about 9 to about 25 amino acid residues and (b) bearing one of the class II HLA motifs set out in Table III.
45. The composition of claim 44, further wherein said peptide is one of those peptides described in Table XVIII or XIX.
46. A composition that comprises an isolated nucleic acid sequence that encodes one of the peptides set out in Tables VI-XIX or XXI or XXIII.
47. A composition that comprises at least two peptides at least one of said at least two peptides selected from Tables VI-XIX or XXI or XXIII.
48. A composition of claim 47 wherein two or more of the at least two peptides are depicted in Tables VI-XIX or XXI or XXIII.
49. A composition that comprises at least one nucleic acid sequence, that encodes the peptides of claim 47.
50. The composition of claim 47 wherein each of said at least two peptides are encoded by a nucleic acid sequence, wherein each of the nucleic acid sequences are located on a single vector.
51. A peptide composition of less than 100 amino acid residues, said composition comprising an epitope useful for inducing an immune response against hepatitis B virus (HBV) said epitope having at least one of the amino acid sequences set out in Table XXIII.
52. A method for inducing a cytotoxic T cell response to HBV in a mammal comprising administering to said mammal at least one peptide from Tables VI to XIX or Table XXI.
53. A vaccine for treating HBV infection that induces a protective immune response, wherein said vaccine comprises at least one peptide selected from Tables VI to Table XIX or Table XXI in a pharmaceutically acceptable carrier.
54. A vaccine for preventing HBV infection that induces a protective immune response, wherein said vaccine comprises at least one peptide selected from Tables VI to XIX or Table XXI in a pharmaceutically acceptable carrier.
55. A method for inducing a cytotoxic T cell response to HBV in a mammal, comprising administering to said mammal a nucleic acid sequence encoding a peptide selected from Tables VI to XIX or Table XXI.
56. A kit for a vaccine for treating or preventing HBV infection, wherein the vaccine induces a protective immune response, said vaccine comprising at least one peptide selected from Tables VI to XIX or Table XXI in a pharmaceutically acceptable carrier and instructions for administration to a patient.
57. A method for monitoring an immune response to HBV or an epitope thereof in a patient having a known HLA-type, the method comprising incubating a T lymphocyte sample from the patient with a peptide selected from Tables VI to XIX or Table XXI, which peptide binds a motif corresponding to at least one HLA allele present in said patient, and detecting the presence of a T lymphocyte that recognizes the peptide.
58. The method of claim 57, wherein the peptide comprises a tetrameric complex.
US12/535,966 1992-01-29 2009-08-05 Inducing Cellular Immune Responses to Hepatitis B Virus Using Peptide and Nucleic Acid Compositions Abandoned US20100068228A1 (en)

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US15918493A 1993-11-29 1993-11-29
US08/159,339 US6037135A (en) 1992-08-07 1993-11-29 Methods for making HLA binding peptides and their uses
US08/197,484 US6419931B1 (en) 1991-08-26 1994-02-16 Compositions and methods for eliciting CTL immunity
US20571394A 1994-03-04 1994-03-04
US08/344,824 US20030152580A1 (en) 1994-07-21 1994-11-23 Hla binding peptides and their uses
US34761094A 1994-12-01 1994-12-01
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US82036097A 1997-03-12 1997-03-12
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US09/189,702 US7252829B1 (en) 1998-06-17 1998-11-10 HLA binding peptides and their uses
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WO2015187009A1 (en) * 2014-06-02 2015-12-10 Isa Pharmaceuticals B.V. Synthetic long peptides (slp) for therapeutic vaccination against hepatitis b virus infection
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